Evaluation of DNA Quality of Beer Ingredients Anna Ramberg131019/FULLTEXT01.pdfresponsible for making beer [3]. Ale yeast is usually used for fermentation at temperatures above 15°C

UPPSALA UNIVERSITY

Final Thesis 10 credit points Level C

Department of Medical Biochemistry

and Microbiology

Spring term 2006

Evaluation of DNA Quality of Beer Ingredients

Anna Ramberg

Supervisor: Dr. Leen Van Houdt

Campus Gildestraat

KaHo Sint-Lieven

Gent, Belgium

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ABSTRACT

The project aim is to determine if good quality DNA can be extracted from barley, malt and

hop, ingredients used in beer brewing. Good quality DNA is important in DNA fingerprinting

techniques which can be used for identification of ingredients. The 3 methods tested are the

cetyltrimethylammonium bromide (CTAB) method, QIAGEN DNeasy Plant Mini Kit and

Meyer’s method as published in 1996 with QIAGEN DNeasy Plant Mini Kit in combination.

To evaluate the DNA quality after extraction we used 3 different techniques:

(i) spectrophotometry to estimate purity by using the ratio A260/A280; (ii) agarose gel

electrophoresis after DNA extraction to determine the success of the extraction and evaluate

the amount of high molecular weight DNA and degradation; and (iii) the polymerase chain

reaction with 4 different primer pairs, together with agarose gel electrophoresis, to determine

if the extracted DNA could be used in downstream applications, see the effect of inhibitors

and estimate the fragmentisation of the DNA. The results achieved using the above mentioned

methods were then used to evaluate the success of each of the extraction methods in their

function of extracting high quality DNA from barley, malt and hop as well as determining

whether the treatment of the ingredients has an effect on the DNA quality.

Key words: CTAB; DNeasy Plant Mini Spin Column; Meyer; Polymerase Chain Reaction;

Malt; Barley; Hop.

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TABLE OF CONTENTS

ABSTRACT………………………………………………………………….Page 2

INTRODUCTION…………………………………………………………...Page 5

Table 1 Malt Types………………….………………………………..Page 7

Table 2 Hop Products……………………….……………………......Page 8

MATERIALS AND METHODS……………………………………………Page 12

1. DNA EXTRACTION METHODS………………………………..Page 12

1.1 Grinding of samples……………………………………...Page 12

1.2 CTAB method…………………………………………….Page 12

1.3 QIAGEN DNeasy Plant Mini Kit………………………..Page 13

1.4 Meyer’s method combined with

QIAGEN DNeasy Plant Mini Kit………………………..Page 15

2. QUALITY ASSESMENT OF DNA…………………………...….Page 15

2.1 Spectrophotometry……………………………………….Page 15

2.2 Agarose gel electrophoresis…………………………...…Page 16

2.2.1 To determine success of DNA extraction……..…Page 16

2.2.2 To determine results of PCR primer pair

PLANT 1 and PLANT 2......................................Page 17

2.2.3 To determine results of the PCR primer pair

rbcL-CRAw Forward and Reverse…………….Page 17

2.2.4 To determine results of the PCR primer pair

combinations with HOR primers………………Page 18

2.3 Polymerase Chain Reaction (PCR)…………………...…Page 18

2.3.1 PCR with plant-specific primers

PLANT 1 and PLANT 2……………………..…Page 18

2.3.2 PCR with plant-specific primers

rbcL-CRAw Forward and Reverse…………….Page 19

2.3.3 PCR with species-specific primers

HOR 1B and HOR 2……………………………Page 20

2.3.4 PCR with increase of amplicon size

using HOR primer pairs…………………...…...Page 22

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RESULTS……………………………………………………………………….Page 23

1. DNA EXTRACTION RESULTS………………………………...….Page 23

Table 7 Scoring DNA

Series 1: Tobacco leaf and Pilot samples…………………………….Page 24

Table 8 Scoring DNA

Series 2: 9 types of malt and 1 barley variety………………………..Page 24

Table 9 Scoring DNA

Series 3: 4 types of hop products………………………………...…...Page 24

2. PCR RESULTS…………………………………………….…......….Page 25

Table 10 Scoring PCR results

Series 1: Tobacco leaf and Pilot samples…….…………………….…Page 25


Series 2: 9 types of malt and 1 barley variety……….………………..Page 26


Series 3: 4 types of hop products………………...………………...…Page28

3. SPECTROPHOTOMETRY RESULTS……………………………...Page 29

Table 13 Scoring spectrophotometric results

Series 1: Tobacco leaf and Pilot samples…………………………….Page 29

Table 14 Scoring spectrophotometric results

Series 2: 9 types of malt and 1 barley variety………………………..Page 30

Table 15 Scoring spectrophotometry results

Series 3: 4 types of hop products………………………..…………...Page 31

Table 16 Spectrophotometric data……...……………………………Page 32

Table 17 Summary…………………………….……………………..Page 34

DISCUSSION……………………………………………………………......….Page 34

ACKNOWLEDGEMENTS………………………………………...…………..Page 44

REFERENCES………………………………………………………...………..Page 44

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INTRODUCTION

The oldest proven records of brewing are about 6 000 years old and come from the Sumerians

who lived in what is modern day Iraq. Later, there are records from the Babylonians in the

same area, the Egyptians and later still the Greeks and Romans. In ancient times beer was

drunk with drinking straws to filter out the bitter brewing residue. The Babylonians

established a law to determine the daily beer ratio for an individual which was dependent on

the person’s social standing, with the upper classes being allowed the larger volume. Beer was

thought to contain a spirit or god since drinking it “possessed” the drinker. The Romans

considered beer the drink of the common man and was held in lower regard to wine. Later in

the Finnish poetic saga Kalewala 400 verses are devoted to beer, but only 200 to the creation

of the earth [1].

Many of the processes and practises of beer-brewing have been handed down from generation

to generation of brewers for centuries, even millennia. The traditional raw materials for

brewing beer are water, malt (a product of barley), hops and yeast. To brew one volume of

beer between 4 and 12 volumes of water is used [2].

The malting of barley (Horduem vulgare) changes the physical and chemical structure of

the grain. Malt is an easily ground product and is ideal for brewing beer, as malted barley has

a high complement of enzymes, which convert starch into simple sugars and proteins, which

are needed for yeast nutrition. Malting involves converting insoluble starch to soluble starch,

reducing complex proteins, generating nutrients for yeast development and development of

enzymes. It involves 3 main processes: steeping, germination and kilning.

Steeping means mixing the barley kernels with water to raise the moisture level and

activate the metabolic processes. The water is replaced several times to maintain a high level

of oxygen-saturation and the grains are turned to maximize their oxygen uptake. The process

is complete when the white tips of the rootlets emerge (chitting). The wet barley is germinated

by maintaining a suitable humidity and temperature. During malt kilning the green malt is

dried in a kiln at different temperatures. The temperature determines the colour of the malt

and the amount of enzymes that survive which in turn determines the type of beer that will be

produced.

Hop comes as whole, kiln-dried cones, powdered hop and hop pellets (powdered hops

packed into pellets) among other forms. Hops are a minor beer ingredient used for bittering,

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flavouring and aroma-enhancing of the beer. It also inhibits the growth of gram-negative

bacteria. It contains resins, oils and phenols among other constituents. Only one species is

used for brewing, Humulus lupulus, but there are several varieties of it used [3]. The wide

variation of chemical profiles found among hop cultivars provides the brewer with the

flexibility to give specific aroma and flavour characteristics to the beer produced [4].

Yeast is a single cell organism that reproduces by budding. This fungus, Saccharomyces

cerevisiae, is responsible for converting fermentable sugars to alcohol and is therefore largely

responsible for making beer [3]. Ale yeast is usually used for fermentation at temperatures

above 15°C while lager yeast is used at fermentation temperatures below 17°C [2].

The malt is milled to split the husk and expose the starchy endosperm for milling and allow

efficient extraction and filtration of the wort. Mashing is the process of converting starch,

from milled malt and other solid adjuncts, into fermentable and unfermentable sugars to

produce wort of the desired composition. Milled malt and other solid adjuncts are mixed with

water at set temperatures and volumes to continue the biochemical changes initiated during

the malting process. Wort separation separates the liquid extract, the wort, from the solids

which are no longer needed. Clear wort is then conditioned by boiling. This stabilises the wort

and extracts such as desirable components from the hops. Here sterilization, destruction of

enzymes, colour development et cetera is done. The wort is then cooled in preparation for

yeast addition and fermentation. Oxygenation to support yeast growth occurs. After this

fermentation and the steps leading to bottling occur [3].

The overall project aim is to isolate DNA from particular ingredients in food or feed that can

be used for DNA-fingerprinting. DNA-fingerprinting techniques such as Random Amplified

Polymorphic DNA (RAPD), Simple Sequence Repeat Polymorphism (SSR) and Amplified

Fragment Length Polymorphism (AFLP) are used to identify and characterise the ingredients

in a specific foodstuff or feed [5]. The quality and authenticity of food products is important

to the modern consumer. Also in the European Community the traceability of the origin,

quality and authenticity of food products is of growing importance. Several European

countries today require the labelling of genetically modified organisms (GMO’s) in food. This

has led to pressure being put on authorities and producers to check food products for accurate

labelling for economic, religious and health reasons. As a consequence, it has become

necessary to develop reliable techniques to trace and label food correctly.

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A concrete example is the fingerprinting of ingredients in a ground meat mix that claims to

contain no pork. Using pork specific primers, the meat can be checked to indeed contain no

pork [6]. The suitability of isolated DNA as an analyte for a detection or characterisation

technique depends on the amount or concentration, purity and integrity; each of these can be

affected by the sample matrix and the extraction method [7]. The treatments of the foodstuffs

can affect the DNA quality and quantity. For example, heat exposure is known to fragmentise

high molecular weight DNA, and PCR-inhibitors may be added during food processing. The

DNA extraction method can also have a big influence on the degradation of the DNA, the

yield and the PCR amplification efficiency. The extracted DNA can, in addition to its use in

identification studies, also be used in detection of GMO’s in food or feed. In recent years

nucleic acids have become an important tool in food analysis to discriminate between

genetically modified and unmodified foodstuffs, which is best achieved at DNA level [8].

During the first year of the project, which is the part I was involved in and will present

here, the goals are to see whether the ingredients we have chosen are appropriate for DNA-

isolation and whether the treatment of the ingredients affects the results, what kind of

extraction methods are the most effective and what the DNA-quality is after extraction. The

ingredients we chose to use are beer ingredients:

(1) 1 variety of barley (Franse Esterelle), a positive control as it is untreated plant material

(2) 9 types of malt, (treated with different end temperatures in the kilning process) to check

the quality and quantity of the extracted DNA depending on the treatment (see Table 1)

(3) 4 different forms of hop products, to check whether hop processing has an influence on

DNA extractability and quality (see Table 2)

Table 1: Malt types

Malt Type End Temperature Original Barley Type

Pilsmout 80°C Franse Scarlett

Ambermout 90°C Franse Scarlett

Cara 20 107°C Franse Optic

Cara 120 132°C Franse Esterelle

Cara 300 145°C Franse Esterelle

MRoost 50 180°C Franse Prestige P.V.

MRoost 450 215°C Franse Esterelle



The different malt types were supplied by Mouterij Dingemans, Belgium

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Table 2: Hop products

Hop Grower/Distributor Variety Crop

Compressed Hop flowers

(BOTANIX)

T.G. Redsell LTD East Kent Goldings 2000

Bale 4072

Hop powder pellets T90 Naudts Magnum 2001

Pre-isomerised Hop

pellets (Sabex:Zambian

Breweries, Lusaka)

Joh. Barth Magnum 2001

Supercritical CO2-extract Magnum 2004

The different hop products were provided by the Laboratory of Enzyme and Brewing Technology at KaHo Sint-Lieven in Gent.

We used tobacco leaf as an experimental control for the different DNA-extraction procedures

as it should give good quality DNA and serves as a positive control. Further, a pilot

experiment was done using one type of powdered hop pellets, one type of malt and one barley

variety, all supplied by the school brewery (not included in Tables 1 and 2), to adapt the

extraction protocols. The food ingredients chosen were beer ingredients because the school

started as a brewery school, several breweries have expressed an interest in the results after

having been informed of the project and because ongoing research at KaHo Sint-Lieven on

brewing technology that made that several of the ingredients are readily available.

We used 3 different DNA-extraction methods:

1. Cetyltrimethylammonium bromide (CTAB) method

2. QIAGEN DNeasy Plant Mini Kit

3. Meyer’s method as published in 1996 and QIAGEN DNeasy® Plant Mini Kit in

combination

To check the quality of the DNA extracted we used:

1. Spectrophotometry

2. Agarose gel electrophoresis

3. Polymerase Chain Reaction (PCR)

-with plant-specific primers (PLANT 1 and 2, rbcL-CRAw Forward and Reverse)

-with barley-specific primers (HOR 1B and HOR 2)

-with increasing size of amplicon (HOR 1B and HOR 2D)

after which the PCR-product was analysed using agarose gel electrophoresis.

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Food matrices are considered suboptimal matrices for DNA-extraction and this affects the

protocols and procedures chosen for the process. Extraction and purification procedures vary

depending on the starting matrix and the use intended for the extracted DNA. To get DNA

from seeds the polysaccharides and proteins need to be removed.

All procedures should:

-disrupt the matrix containing the nucleic acid

-inhibit and/or inactivate nucleases

-solubilise the nucleic acid

-preserve the nucleic acid integrity

This is usually achieved by:

-physical disruption of the sample e.g. grinding

-chemical treatments e.g. detergent lysis

-enzymatic treatments e.g. proteinase K [9]

The cetyltrimethylammonium bromide (CTAB) protocol was first developed by Murray and

Thompson in 1980 [10]. This method is suitable for extraction and purification of DNA from

plants and plant derived foods and is very effective in eliminating polysaccharides and

polyphenolic compounds that can affect DNA purity and quality. CTAB is an ionic detergent

that lyses the plant cells and forms an insoluble complex with nucleic acids in low salt

environments. Polysaccharides, phenols and other contaminants can be washed away. Raising

the salt concentration solubilises the DNA and it can then be precipitated using ethanol or

isopropanol [11].

Advantages:

-can be applied to most plant derived food and feed matrices

-several variants have been developed to adapt the method to particular matrices

-relatively low cost

Disadvantages:

-procedure is rather laborious and time consuming

-with some matrices inhibitors of enzymatic reactions might be co-purified [9]

The DNeasy® Plant Mini Kit provides a fast and easy way to isolate plant DNA. The DNeasy

Spin Column procedure uses a silicon column-based technique that gives pure total DNA in a

short time. The DNeasy membrane removes PCR-inhibitors. The purification doesn’t use

phenol or chloroform extraction or alcohol precipitation and minimises handling. The sample

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is first mechanically disrupted and then lysed by adding the lysis buffer. RNase A is added to

digest RNA. Next, proteins and polysaccharides are precipitated and together with cell debris,

removed by centrifugation through the QIAshredder Column. Binding buffer and ethanol is

added to the cleared lysate which promotes the binding of DNA to the DNeasy Spin Column.

After washing, the DNA can be eluted using a low-salt buffer (AE buffer) or water. The

purified DNA is mostly fragments of 20-25 kb but can be up to 40 kb in size and shows high

amplification efficiency in PCR.

Advantages:

-fast and simple to use

-high quality DNA

-involves no chloroform which is toxic if inhaled

-extensive quality control and warranties provided by QIAGEN

-technical assistance available

Disadvantages:

-the ingredients of the buffers are unknown to the user

-the lysis step is not always efficient on some types of plant material [12]

DNA-extraction using the method developed by Meyer in 1996 [13] in combination with the

DNeasy Plant Mini Kit combines a powerful lysis buffer with a silicon column. The lysis step

is more efficient than in the DNeasy method and, since lysis of the cells is a crucial step in

order to obtain DNA, this method is useful with more resistant cell types. It is combined with

DNeasy since DNeasy is known to give pure DNA.

Spectrophotometry is used to determine the purity of the extracted DNA. Nucleic acids absorb

light at 260nm and the amount of light absorbed can be used to calculate the purity and

amount of DNA. By using a spectrophotometer that emits a light at 260nm that passes

through the sample, the concentration of DNA in the sample can be determined. The more

light is absorbed the more nucleic acid is present in the sample [14]. Interference by

contaminants is calculated by using a ratio. Since proteins absorb at 280nm, the ratio of

absorbance A of a sample at 260nm and the absorbance A at 280nm can be used to estimate

the purity of the DNA-sample. Pure DNA should give a ratio of above 1.8. A A260/A280 ratio

below 1.8 indicates that the sample is contaminated with proteins and/or aromatic substances

(e.g. phenols). A A260/A280 ratio above 2.0 indicates a possible contamination with RNA [15].

Absorption at 230nm indicates contamination of the sample by carbohydrates, peptides,

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phenols and aromatic compounds. The ratio A260/A230 should be about 2.2 for pure DNA

samples. Absorption at 320nm can be due to a precipitate or particles in the sample or dirty or

damaged cuvettes [11].

Gel electrophoresis is a method that separates macromolecules based on size, electric charge

and other physical properties. This is done by applying a voltage between electrodes at each

end of the gel. The frictional force of the gel material acts as a molecular sieve and separates

molecules by size. Large molecules or fragments will migrate slower and small ones will

migrate faster through the gel. The net charge of the molecule determines whether the

migration is towards the anode or the cathode. DNA molecules are negatively charged as a

result of the phosphate groups (PO4¯) which cause the DNA to migrate towards the anode.

Agarose gel electrophoresis is a standard method for separating, identifying and purifying

DNA-fragments. The location of the DNA in the gel is determined by staining the gel with

ethidium bromide, a fluorescent intercalating dye. A DNA marker can be used to estimate the

concentration or size (number of base pairs) of the DNA bands produced. This technique is

simple and relatively quick and allows to check DNA integrity, degradation and possible

RNA contamination [16].

Because DNA is more stable than other molecules, e.g. proteins, to the chemical and physical

treatments during food processing, tests based on DNA identification are useful for

authenticating the species used in foodstuffs production and determining traceability of an

ingredient. The method used most today is the polymerase chain reaction (PCR) which is

based on the natural DNA-replication mechanism [17]. The technique consists of repetitive

cycles of denaturation of the DNA to single stranded DNA using high temperatures; annealing

of 2 oligonucleotides, primers, to the target sequence; and the extension of the DNA chain by

adding nucleotides and using a thermostable DNA polymerase. The primers are short DNA

sequences, different to each other and complementary to the sequence they are to recognise.

Primers can be designed and chosen to target specific genes. Since after each cycle the new

DNA can also act as templates in the next cycle the amplification of the DNA is exponential,

so a relatively small amount of DNA can be amplified and analysed. DNA quality can be

checked using PCR amplification since positive results will indicate that there are no or low

levels of PCR-inhibitors in the DNA extract. Also the amplicon size used can determine if the

DNA integrity is sufficient for amplication [18]

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MATERIALS AND METHODS

1. DNA EXTRACTION METHODS

Each of the three extraction methods requires 100mg starting material and the extracted DNA

in each case is dissolved in 100µl AE-buffer (component of the QIAGEN DNeasy Plant Mini

Kit) which makes them easy to compare.

1.1 Grinding of samples

Weigh 2g of the malt seeds and the hop products and 6g of the barley seeds in disposable

weighing dishes. Avoid contamination by only having one sample at a time out in the open.

Grind the malt and the hop products to a fine powder using a mortar and pestle. Clean these

carefully before use with hot water and detergent, rinse with deionised water, wipe with

DNAerase™ (DNA Contaminant Removal Solution, Catalogue number 821805, MP

Biomedicals Inc, www.mpbio.com), wipe with deionised water and dry. Work in a separate

room from the one where extraction and PCR will be done. Cover the work area with plastic

film for easy cleaning between samples. Put the powder in a labelled 15ml tube and store at

room temperature in a dry place until use. The barley has very hard kernels and is difficult to

grind by hand. Use a kitchen grinding machine or similar. We used a Rondo 300 from SEB

that was bought in an ordinary appliance store. Even using such a machine there will be a lot

of material that is too big for extraction, hence the extra material weighed. Try to separate the

powder from the debris as well as possible and put the powder in a labelled 15ml tube and

store at room temperature for future use.

1.2 CTAB method

Weigh 100mg of a ground sample in a 1.5ml microcentrifuge tube. Each sample should be in

duplicate. Add 300µl sterile deionised water and mix by vortexing. Add 500µl CTAB-buffer

(Mix 4g CTAB, 16.4g NaCl, 3.15g Tris-HCl and 1.5g Na2EDTA. Add 100ml deionised water

and adjust pH to a value of 8.0 with 1M NaOH or 1M HCl. Fill up to 200ml with deionised

water and autoclave. Final concentrations are 2% CTAB, 1.4M NaCl, 0.1M Tris-HCl and

20mM Na2EDTA). Mix by vortexing. Adjust the volumes until a viscous mixture is obtained.

Add 20µl Proteinase K, concentration 20mg/ml, (Catalogue number P8102S, New England

BioLabs Inc. www.neb.com), shake and incubate at 65°C for 60 minutes. Add 2µl RNase A,

concentration 100mg/ml, (Catalogue number 19101, QIAGEN, www.qiagen.com ), shake and

http://www.mpbio.com/

http://www.neb.com/

http://www.qiagen.com/

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incubate for 10 minutes at 65°C. Centrifuge for 10 minutes at 16 000xg (centrifuge type 5415

D, Eppendorf, www.eppendorf.com). Transfer the supernatant to a microcentrifuge tube

containing 500µl chloroform/isoamylalcohol (24/1) and shake for 30 seconds. Centrifuge for

10 minutes at 16 000xg. Transfer 500µl of the upper layer to a new microcentrifuge tube

containing 500µl chloroform/isoamylalcohol and shake for 30 seconds. Centrifuge for 5

minutes at 16 000xg. Transfer the upper layer to a new microcentrifuge tube (typically about

400µl can be safely transferred). Add 2 volumes of CTAB precipitation solution (Mix 1g

CTAB and 0.5g NaCl. Add 100ml deionised water and adjust pH to 8.0 with 0.1M NaOH.

Fill up to 200ml and autoclave. Final concentrations are 0.5% CTAB and 0.04M NaCl) and

mix by inverting. Incubate at room temperature for 60 minutes. Centrifuge for 5 minutes at

16 000xg and discard the supernatant. Dissolve the precipitate in 350µl 1.2M NaCl and add

350µl chloroform/isoamylalcohol and shake for 30seconds. Centrifuge for 10 minutes at

16 000xg. Transfer the upper layer to a new microcentrifuge tube (typically about 300µl can

be safely transferred). Add 0.6 volumes of isopropanol and shake for 30 seconds. Centrifuge

for 10 minutes at 16 000xg. Discard the supernatant by pipetting. Add 500µl of 70% ethanol

solution and shake carefully. Centrifuge for 10 minutes at 16 000xg. Discard the supernatant

with a micropipette. Centrifuge for a few seconds and discard the remaining supernatant. Air-

dry the pellets and re-dissolve the DNA in 100µl AE-buffer (see DNeasy Plant Mini Kit).

Label and store the samples at -20°C until needed.

This protocol is adapted from the method described in The Analysis of Food Samples for

the Presence of Genetically Modified Organisms, Session 4, Extraction and Purification of

DNA by M. Somma for the European Commission Joint Research Centre (reference 11). We

have standardised the incubation times, used chloroform mixed with isoamylalcohol (24/1) to

reduce evaporation and added a short centrifugation step to eliminate all ethanol as it is a

PCR-inhibitor. Experimental: when extracting DNA from tobacco leaf, 300µl of water was

not added in the first step. Instead the leaf was crushed with a mini-pestle with 500µl CTAB-

buffer. After 50 minutes of incubation 150µl water was added to the mixture to increase the

liquidity of the sample.

1.3 DNeasy® Plant Mini Kit (Catalogue number 69106, QIAGEN, www.qiagen.com)

Weigh 100mg of a ground sample in a 1.5ml microcentrifuge tube in duplicate. Add 400µl of

Buffer AP1 and 4µl of RNase A, concentration 100g/ml, and vortex vigorously until a

homogenous mixture is obtained. Incubate for 30 minutes at 65°C. Mix 2-3 times during

incubation by inverting the tube. This step lyses the cells. Add 130µl of Buffer AP2 to the

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tube, mix and incubate on ice for 5 minutes. This step precipitates detergents, proteins and

polysaccharides. Centrifuge for 15 minutes at 16 100xg. Apply the supernatant to the

QIAshredder Mini Spin Column placed in a 2ml collection tube (supplied with kit) and

centrifuge for 2 minutes at 16 100xg. Transfer the flow-through fraction to a new tube (not

supplied) without disturbing the cell-debris pellet (typically between 300-500µl is recovered,

note the volume). Add 1.5 volumes of Buffer AP3/E (ensure that ethanol has been added to

Buffer AP3/E according to instructions in kit) to the cleared lysate and mix immediately.

Apply 650µl of the mixture, including any precipitate which may have formed, to the DNeasy

Mini Spin Column sitting in a 2ml collection tube (supplied with kit). Centrifuge for 1 minute

at 8 000xg and discard the flow-through. Reuse the collection tube in the next step. Repeat the

procedure using the rest of the sample. Discard the flow-through and the collection tube.

Place the DNeasy Mini Spin Column in a new 2 ml collection tube (supplied with kit), add

500µl Buffer AW (ensure ethanol is added to Buffer AW according to instructions in kit) to

the DNeasy Mini Spin Column and centrifuge for 1 minute at 8 000xg. Discard the flow-

through and reuse the collection tube in the next step. Add 500µl of Buffer AW to the DNeasy

Mini Spin Column and centrifuge for 2 minutes at 16 100xg to dry the membrane. Ethanol is

a PCR-inhibitor so it is important that no residual ethanol is left. Discard the flow-through and

the collection tube. Transfer the DNeasy Mini Spin Column to a 1.5ml microcentrifuge tube

(not supplied) and pipette 50 µl of Buffer AE directly on to the DNeasy membrane. Incubate

for 5 minutes at room temperature (15-25°C) and then centrifuge for 1 minute at 8 000xg to

elute. Repeat the previous step once to obtain a final volume of 100µl. Label and store the

samples at -20°C for future use.

The original protocol was developed by QIAGEN for the DNeasy Plant Mini Kit in January

2004 and can be found in their handbook. We have changed the following to ensure

acceptable results from the samples we used: the incubation time is increased from 10 to 30

minutes after adding Buffer AP1 and RNase A. The optional centrifugation step was included,

but for 15 minutes instead of 5 minutes to get a clear supernatant. Our microcentrifuge had a

maximum speed of 16 100xg. We did not mix by pipetting after adding Buffer AP3/E to avoid

contamination, instead we shook the tubes. We increased the centrifugation speed from

6000xg to 8000xg to ensure that washing and elution steps were completed. We eluted with

50µl x 2 to increase the final DNA concentration in the eluate. Hops needed 800µl of Buffer

AP1 to homogenise. Experimental: When extracting DNA from tobacco leaf the material was

crushed with a mini-pestle together with Buffer AP1.

15

1.4 Meyer´s method combined with QIAGEN DNeasy Plant Mini Kit

Weigh 100mg of a ground sample in a 1.5ml microcentrifuge tube in duplicate. Add 430µl

extraction buffer (10mM Tris pH 8.0, 150mM NaCl, 2mM EDTA and 1% SDS) and 50µl 5M

guanidine hydrochloride. Vortex vigorously until the mixture is viscous. If necessary, adjust

volumes until a viscous mixture is obtained. Add 20µl Proteinase K, concentration 20mg/ml,

and mix by inverting. Incubate for 3 hours at 57°C. Homogenise every 30 minutes by

inverting the tube. Centrifuge for 15 minutes at 14 500xg. Transfer the lysate to the

QIAshredder Mini Spin Column and centrifuge for 2 minutes at 16 100xg. Transfer the flow-

through to a new tube (not supplied) without disturbing the pellet. Note the volume recovered.

Add 1.5 volumes of Buffer AP3/E to the transferred lysate and mix well immediately without

vortex. Apply 650µl of the lysate, including any precipitate which may have formed, to the

DNeasy Mini Spin Column sitting in a 2ml collection tube (supplied with DNeasy Plant Mini

Kit). Centrifuge for 1 minute at 8 000xg and discard the flow-through. Repeat the previous

step with remaining sample. Discard the flow-through and collection tube. Place the DNeasy

Mini Spin Column in a new 2ml collection tube (supplied with kit). Add 500µl Buffer AW to

the DNeasy Mini Spin Column and centrifuge for 1 minute for 8 000xg. Discard the flow-

through and reuse the collection tube in the next step. Add 500µl Buffer AW to the DNeasy

Mini Spin Column and centrifuge for 2 minutes at 16 100xg to dry the membrane. Transfer

the DNeasy Mini Spin Column to a new 1.5ml microcentrifuge tube and add 50µl of Buffer

AE directly on to the DNeasy Mini Spin Column membrane. Incubate for 5 minutes at room

temperature and centrifuge for 1 minute at 8 000xg to elute. Repeat the previous step once to

obtain a final volume of 100µl. Label and store the tubes at -20°C for future use.

Hops required double the amounts above to homogenise (860µl extraction buffer and 100µl

guanidine hydrochloride). Experimental: when extracting DNA from tobacco leaf 215µl

extraction buffer, 25µl guanidine hydrochloride and 10µl 20mg/ml Proteinase K was used.

2. QUALITY ASSESMENT OF EXTRACTED DNA

2.1 Spectrophotometry

Mix 7µl of the DNA sample with 63µl pure HPLC-grade water to make a 1/10 dilution with a

total volume of 70µl in a 1.5ml eppendorf tube. Mix well and centrifuge for a few seconds to

concentrate the fluid in the bottom of the tube. Pipette 60µl of the dilution in to the bottom of

a plastic disposable cuvette (Single sealed UVette®, Order number 0030 106.300, Eppendorf,

www.eppendorf.com). Avoid air bubbles as they will interfere with the light ray and give


16

false results. Place a cuvette containing the blank, 60µl pure water, in position in the

spectrophotometer (BioPhotometer No.6131 00389, Eppendorf, www.eppendorf.com). Make

sure to place it so that the clear sides are in line with the direction of the light ray. Switch the

photometer on and programme it to measure double stranded DNA. Measure the blank by

pushing the button marked “Blank” and make sure the result reads 0. Place the cuvette

containing the diluted sample in position in the photometer and press the “Sample” button to

measure. The instrument will show its readings for absorbance at 230nm, 260nm, 280nm and

320nm as well as the ratio of the absorbance A260nm/A280nm and A260nm/A230nm. It will also

show the concentration of DNA in the sample in µg/ml. Note all the results down.

Since we dissolved all samples in AE-buffer, a 1/10 dilution of AE-buffer was also

measured to get the background absorbance compared to the blank which was HPLC-grade

water.

2.2 Agarose gel electrophoresis

2.2.1 To determine success of DNA extraction

Make a 1% agarose gel with 1.5g agarose and 150ml 1x Tris/Acetic acid/EDTA (TAE) buffer

(Mix 20ml 50x TAE stock solution (Catalogue number 161-0773, Bio-Rad Laboratories,

www.bio-rad.com) with 980ml deionised water to make a 1x solution with the concentrations

40mM Tris, 20mM acetic acid and 1mM EDTA and with a pH of 8.3). Microwave for 5

minutes at 500W or until the agarose powder is completely melted. Cool under running water

and pour carefully in to the gel casting system with the 2 combs in place. Ensure that no air

bubbles are present and allow the gel to set. After it has set, remove the combs carefully and

cover the gel with 1x TAE-buffer. Turn the gel so that it is correctly oriented with respect to

the anode and cathode. Make a λ-DNA (λ-DNA 250ng/µl, Catalogue no 10 745 782 001,

Roche Diagnostics, www.roche.com) stock with a concentration of 25ng/µl by mixing 10µl λ-

DNA 250ng/µl stock with 90µl sterile water. Make a dilution series from the 25ng/µl stock by

mixing increasing volumes of the stock with decreasing volumes of sterile water to a final

volume of 10µl (50ng = 2µl 25ng/µl λ-DNA stock + 8µl H2O, 100ng = 4µl 25ng/µl λ-DNA

stock + 6µl H2O, 150ng = 6µl 25ng/µl λ-DNA stock + 4µl H2O, 200ng = 8µl 25ng/µl λ-DNA

stock + 2µl H2O). This series can be used to estimate the concentration of the extracted DNA

by comparing the band intensities. Add 2µl loading buffer (50% glycerol, 50% sterile water

and a spatula tip each of xylene cyanol and bromophenol blue) to 10µl of each sample from


http://www.bio-rad.com/

http://www.roche.com/

17

the DNA-extraction as well as to the λ-DNA dilution series to obtain a final volume of 12µl

in each case. Load 11.5µl of each dilution and sample in a separate well in the gel. Make a

loading scheme to record the sample contained in a well. Run at 100V, 400mA for 1.5 hours.

Divide the gel in 2 using a scalpel to make staining and photographing easier. Stain in an

ethidium bromide bath (1xTAE buffer + 1 drop ethidium bromide solution (10mg/ml,

catalogue number 161-0433, Bio-Rad Laboratories, www.bio-rad.com) per 100ml buffer) for

45 minutes and view the results using UV-light. Photograph the gel to record the results.

2.2.2 To determine results of the PCR primer pair PLANT 1 and PLANT 2

Make a 1.5% agarose gel with 2.25g agarose and 150ml 1x Tris/Acetic acid/EDTA (TAE)

buffer. Microwave for 5 minutes at 500W or until the agarose powder is completely melted.

Cool under running water and pour carefully in to the gel casting system with the 2 combs in

place. Ensure that no air bubbles are present and allow the gel to set. After it has set, remove

the combs carefully and cover the gel with 1x TAE-buffer. Turn the gel so that it is correctly

oriented with respect to the anode and cathode. Add 2µl loading buffer to each PCR tube

containing the samples from the PCR. Load 12µl of each sample in a separate well in the gel.

On each gel load 12µl of a 100 base pair DNA ladder (TrackIt™ 100bp DNA ladder,

0.1ug/ul, Catalogue number 10488-058, Invitrogen Life Technologies, www.invitrogen.com)

to be able to estimate the size of the bands produced after separation. Make a loading scheme

to record the sample contained in a well. Run at 100V, 400mA for 1 hour 15 minutes. Divide

the gel using a scalpel to make staining and photographing easier. Stain in an ethidium

bromide bath for 50 minutes and view the results using UV-light. Photograph the gel to record

the results.

2.2.3 To determine results of the PCR primer pair rbcL-CRAw Forward and Reverse



Cool under running water and pour carefully in to the gel casting system with only the top

comb in place to be able to use the whole gel for the run. Ensure that no air bubbles are

present and allow the gel to set. After it has set, remove the comb carefully and cover the gel

with 1x TAE-buffer. Turn the gel so that it is correctly oriented with respect to the anode and

cathode. Add 2µl loading buffer to each PCR tube containing the samples from the PCR.

Load 12µl of each sample in a separate well in the gel. Load 12µl of a 100 base pair DNA


http://www.invitrogen.com/

18

ladder to be able to estimate the size of the bands produced after separation. Make a loading

scheme to record the sample contained in a well. Run at 100V, 400mA for 2 hours 15

minutes. Stain the gel in an ethidium bromide bath for 1 hour and view the results using UV-

light. Photograph the gel to record the results.

2.2.4 To determine the results of the PCR primer pair combinations with HOR primers



Cool under running water and pour carefully in to the gel casting system with the 2 combs in

place. Ensure that no air bubbles are present and allow the gel to set. After it has set, remove

the combs carefully and cover the gel with 1x TAE-buffer. Turn the gel so that it is correctly

oriented with respect to the anode and cathode. Add 2µl loading buffer to each PCR tube

containing the samples from the PCR. Load 12µl of each sample in a separate well in the gel.

Load 12µl of a 100 base pair DNA ladder to be able to estimate the size of the bands

produced after separation. Make a loading scheme to record the sample contained in a well.

Run at 100V, 400mA for 1 hour 30 minutes. Stain the gel in an ethidium bromide bath for 1

hour and view the results using UV-light. Photograph the gel to record the results.

2.3 Polymerase Chain Reaction (PCR)

To avoid contamination, work in a sterile environment, for example a laminar floor equipped

with UV-light (do not use air flow functions). Wipe all surfaces with DNAerase. Switch on

the UV-light for 15 minutes before use to sterilise the surfaces. Use only filtered tips to avoid

aerosol contamination between samples by the pipette. The pipettes used should be used

exclusively for PCR and should be wiped regularly with DNAerase. Wear powder-free gloves

at all times. Open only one DNA sample at a time. Aliquote pure water in eppendorf tubes

that can be used and thrown away after use. Work on ice at all times to avoid the unintended

and aspecific interactions and products that can occur at room temperature.

2.3.1 PCR with plant-specific primers PLANT 1 and PLANT 2

PLANT 1 and 2 replicate a section of non-coding chloroplast DNA, which is an abundant

target. This amplicon is conserved between plant species and is 500-600 base pairs (bp) in

length. The primer sequences and reaction conditions were kindly provided by Dr. I.

Taverniers at the Institute for Agricultural and Fisheries Research (ILVO),

www.ilvo.vlaanderen.be.

http://www.ilvo.vlaandern.be/

19

Table 3 Primers PLANT 1 and PLANT 2

Primer

Name

Primer

Length

Primer

Sequence

(5ʹ to 3ʹ)

Molecular

weight

µg/mole

Tm: Melting

Temperature (50mM salt conc)

% GC

PLANT 1 20 base pairs CGA AAT CGG

TAG ACG CTA CG 6152.0 49.0°C 55

PLANT 2 20 base pairs GGG GAT AGA

GGG ACT TGA AC 6272.0

49.0°C 55

Dissolve the primers PLANT 1 and PLANT 2 (Catalogue number 10336-022,

Identification number H8080B05 and H8080B06 respectively, Invitrogen Life Technologies,

www.invitrogen.com) to a concentration of 100mM using ultrapure HPLC-grade water.

Calculate and prepare the master mix in an eppendorf tube according to the number of

samples, including 2 nontemplate controls and 2 extra. For one sample, mix 20.15µl pure H2O

HPLC-grade; 2.5µl 10x PCR buffer*; 0.75µl 50mM MgCl2*; 0.25µl 20mM dNTPs**; 0.1µl

each of PLANT 1 and PLANT 2; and 0.15µl of Taq Polymerase* (5 U/µl) (* 500U Taq DNA

Polymerase Combo, Catalogue number 18038067, Invitrogen Life Sciences,

www.invitrogen.com, ** DNA Polymerisation Mix, Catalogue number 27-2094-02, GE

Healthcare Life Sciences, www.gehealthcare.com). The total volume per sample is 24µl.

Distribute 24µl of the master mix to each of the PCR tubes needed. Add 1µl of ultrapure

water to the first and last tube in the sequence as nontemplate controls. Add 1µl of the DNA

samples to the remaining tubes. Close the lids carefully and place the tubes in the PCR

machine (iCycler™ Thermal Cycler, Bio-Rad Laboratories, www.bio-rad.com). Set the

following programme: 1 cycle of initial denaturation at 94°C for 4 minutes, 35 cycles of

amplification with a denaturation step of 30 seconds at 94°C, an annealing step of 30 seconds

at 55°C and an elongation step of 1 minute at 72°C, 1 cycle of final elongation at 72°C for 3

minutes and finally a hold step at 14°C.

2.3.2 PCR with plant-specific primers rbcL-CRAw Forward (F) and rbcL-CRAw Reverse (R)

The rbcL primer pair replicates a coding section of chloroplast DNA of the ribulose

bisphosphate carboxylase Large subunit gene. They are degenerate primers to make them

universal, which means that they have mixed bases of C+T (designation Y) as well as

A+C+G+T (designation N). This is achieved by having the synthesizer deliver an equal

amount of each base at the given base addition. The amplicon length produced is about 106



http://www.gehealthcare.com/


20

base pairs. The primer sequences and reaction conditions were kindly provided by Dr. G.

Berben at the Walloon Agriculture Research Centre (CRA-W), www.cra.wallonie.be.

Table 4 Primers rbcL-CRAw-F and rbcL-CRAw-R

Primer

Name

Primer

Length

Primer

Sequence

(5ʹ to 3ʹ)

Molecular

weight

µg/mole

Tm: Melting


% GC

rbcL-CRAw-

Forward

26 base pairs CTT ACC AGY

CTT GAT CGT TAC

AAA GG

7938.7 52.0°C 44

rbcL-CRAw-

Reverse

26 base pairs CAA AAA GGT

CTA ANG GYT

AAG CTA CA

8009.5 50.0°C 38

Dissolve the primers rbcL-CRAw-F and rbcL-CRAw-R (Catalogue number 10336-022,

Identification number H8080B07 and H8080B08 respectively, Invitrogen Life Technologies,

www.invitrogen.com) to a concentration of 100mM using ultrapure HPLC-grade water.

Calculate and prepare the master mix in an eppendorf tube according to the number of

samples, including 2 nontemplate controls and 2 extra. For one sample, mix 16.725µl pure

H2O HPLC-grade; 2.5µl 10x PCR buffer*; 1.25µl 50mM MgCl2*; 0.25µl 20mM dNTPs**;

0.15µl of rbcL-CRAw-F; 0.225µl of rbcL-CRAw-R; and 0.15µl of Taq Polymerase* (5 U/µl)

(* 500U Taq DNA Polymerase Combo, Catalogue number 18038067, Invitrogen Life

Sciences, www.invitrogen.com, ** DNA Polymerisation Mix, Catalogue number 27-2094-02,

GE Healthcare Life Sciences, www.gehealthcare.com). The total volume per sample is

21.25µl. Distribute 21.25µl of the master mix to each of the PCR tubes needed. Add 3.75µl of

ultrapure water to the first and last tube in the sequence as nontemplate controls. Add 3.75µl

of the DNA samples to the remaining tubes. Close the lids carefully and place the tubes in the

PCR machine (iCycler™ Thermal Cycler, Bio-Rad Laboratories, www.bio-rad.com). Set the

following programme: 1 cycle of initial denaturation at 95°C for 4 minutes, 50 cycles of

amplification with a denaturation step of 15 seconds at 94°C, an annealing step of 15 seconds

at 60°C and an elongation step of 15 seconds at 72°C, 1 cycle of final elongation at 72°C for 5

minutes and finally a hold step at 14°C.

2.3.3. PCR with species-specific primers HOR 1B and HOR 2

These primers are barley specific primers. They amplify the γ-hordein gene that is a single

copy storage protein gene that is nuclear encoded. Plant storage proteins are known to be

http://www.cra.wallonie.be/





21

highly species specific [17]. It gives an indication whether nuclear DNA can be used as

template. The amplicon length produced by this primer pair is 197 base pairs. This PCR is

based on the conditions as described in reference 17. However, changes have been introduced.

Table 5 Primers HOR 1B and HOR 2

Primer

Name

Primer

Length

Primer

Sequence

(5ʹ to 3ʹ)

Molecular

weight

µg/mole

Tm: Melting


% GC

HOR 1B

(forward)

21 base pairs GCA ACA GAG

TTG TCG GGT 6512.2 60.0°C 57

HOR 2 (reverse) 20 base pairs GAC CCT GGA

CGA GCA CAC AT 6097.0 59.3°C 60

Dissolve the primers HOR 1B (designed by Leen Van Houdt using Invitrogen Custom

Primers, Catalogue number 10336-022, Identification number H8928B10, Invitrogen Life

Technologies, www.invitrogen.com) and HOR 2 (Catalogue number 10336-022,

identification number H8080B04, Invitrogen Life Technologies, www.invitrogen.com) to a

concentration of 100mM using ultrapure HPLC-grade water. Calculate and prepare the master

mix in an eppendorf tube according to the number of samples, including 2 nontemplate

controls and 2 extra. For one sample, mix 20.2µl pure H2O HPLC-grade; 2.5µl 10x PCR

buffer*; 0.75µl 50mM MgCl2*; 0.25µl 20mM dNTPs**; 0.075µl of HOR 1B; 0.075µl of

HOR 2; and 0.15µl of Taq Polymerase* (5 U/µl) (* 500U Taq DNA Polymerase Combo,

Catalogue number 18038067, Invitrogen Life Sciences, www.invitrogen.com, ** DNA

Polymerisation Mix, Catalogue number 27-2094-02, GE Healthcare Life Sciences,

www.gehealthcare.com). The total volume per sample is 24µl. Distribute 24µl of the master

mix to each of the PCR tubes needed. Add 1µl of ultrapure water to the first and last tube in

the sequence as nontemplate controls. Add 1µl of the DNA samples to the remaining tubes.

Close the lids carefully and place the tubes in the PCR machine (iCycler™ Thermal Cycler,

Bio-Rad Laboratories, www.bio-rad.com). To screen for a primer annealing temperature for

the primers used, a gradient PCR was performed. The annealing step in the amplification

cycle was programmed by the machine with an 8-step gradient between 57-61°C to find the

optimal temperature. The results showed that 61°C gave the least amount of aspecific

amplification so this temperature was selected as the annealing temperature. Set the following

programme: 1 cycle of initial denaturation at 95°C for 4 minutes, 40 cycles of amplification






22

with a denaturation step of 30 seconds at 95°C, an annealing step of 30 seconds at 61°C and

an elongation step of 45 seconds at 72°C and finally a hold step at 14°C.

2.3.4. PCR with increase of amplicon size using HOR primer pairs

Increasing the amplicon size using species specific primers can help determine how well

conserved the barley DNA is in different malt types that have been treated with different

temperatures during kilning. This can help determine how high the temperature can be before

the DNA is so degraded that PCR can not be done. The larger the amplicon size that can be

replicated, the less the template DNA has been fragmented. We designed primers using the

HOR 1 (forward) and HOR 2 (reverse) primer pair as starting points in the γ-hordein gene.

HOR 1, 1B, 1C and 1D and HOR 2, 2B, 2C and 2D all start at different points on the γ-

hordein gene and can therefore be combined in a number of different ways to give amplicons

with different lengths. We used primer pair HOR 1B + HOR 2 as our first amplicon size (see

section 2.3.3) at 197 base pairs and increased the amplicon size to 645 base pairs by using

primer pair HOR 1B + HOR 2D.

Table 6 Primers HOR 1B and HOR 2D

Primer

Name

Primer

Length

Primer

Sequence

(5ʹ to 3ʹ)

Molecular

weight

µg/mole

Tm: Melting


% GC

HOR 1B

(forward)

21 base pairs GCA ACA GAG

CAG TTG TCG

GGT

6512.2 60.0°C 57

HOR 2D

(reverse)

20 base pairs TCG ACG ACT

AAC ACC GAA GG 6121.0 57.0°C 55

Dissolve the primers HOR 1B and HOR 2D (designed by Leen Van Houdt using Invitrogen

Custom Primers, Catalogue number 10336-022, Identification number H8928B10 and

H8928C03 respectively, Invitrogen Life Technologies, www.invitrogen.com) to a

concentration of 100mM using ultrapure HPLC-grade water. Calculate and prepare the master

mix in an eppendorf tube according to the number of samples, including 2 nontemplate

controls and 2 extra. For one sample, mix 20.2µl pure H2O HPLC-grade; 2.5µl 10x PCR

buffer*; 0.75µl 50mM MgCl2*; 0.25µl 20mM dNTPs**; 0.075µl of HOR 1B; 0.075µl of

HOR 2D; and 0.15µl of Taq Polymerase* (5 U/µl) (* 500U Taq DNA Polymerase Combo,

Catalogue number 18038067, Invitrogen Life Sciences, www.invitrogen.com, ** DNA



23

Polymerisation Mix, Catalogue number 27-2094-02, GE Healthcare Life Sciences,

www.gehealthcare.com). The total volume per sample is 24µl. Distribute 24µl of the master

mix to each of the PCR tubes needed. Add 1µl of ultrapure water to the first and last tube in

the sequence as nontemplate controls. Add 1µl of the DNA samples to the remaining tubes.

Close the lids carefully and place the tubes in the PCR machine (iCycler™ Thermal Cycler,

Bio-Rad Laboratories, www.bio-rad.com). Set the following programme: 1 cycle of initial

denaturation at 95°C for 4 minutes, 40 cycles of amplification with a denaturation step of 30

seconds at 95°C, an annealing step of 30 seconds at 61°C and an elongation step of 45

seconds at 72°C and finally a hold step at 14°C.

RESULTS

1. DNA EXTRACTION RESULTS

DNA was extracted from 3 series of samples in duplicate:

Series 1: 100mg tobacco leaf and 100mg of each of the pilot experiment samples consisting of

one malt type, one barley variety and one hop product.

Series 2: 100mg of each of the 9 malt types in Table 1 and from Franse Esterelle (barley).

Series 3: 100mg and 20mg of each of the hop products in Table 2, except hop extract which

had only 100mg samples.

DNA was extracted using the 3 protocols described: CTAB (1.2), DNeasy (1.3) and Meyer

+ DNeasy (1.4). Agarose gel electrophoresis was done as described earlier and the appearance

of the bands scored according to the amount and integrity of the DNA. The results are

summarised in Tables 8 to 10. The duplicate samples are referred to as 1 and 2.

Legend for tables “Scoring of DNA”

+++ High concentration band

++ Medium concentration band

+ Low concentration band

- Negative sample

S Smeared

D Degraded

HD Highly degraded



24

Table 7 Scoring DNA Series 1: Tobacco leaf and Pilot Experiment Method

Tobacco leaf

Barley Malt Hop pellets

Water

CTAB 1 + +++ S +++ S - -

2 + +++ S +++ S - -

DNeasy 1 +++ + +++ S ++ S -

2 +++ + +++ S ++ S -

Meyer 1 +++ + D ++ S,D - -

2 + + D ++ S,D - -

Table 8 Scoring DNA Series 2: 9 types of malt and 1 barley variety

80°C 90°C 107°C 132°C 145°C 180°C 215°C 225°C 250°C

Method

Pilsmout Ambermout Cara 20 Cara 120 Cara 300 MRoost 50

MRoost 450

MRoost 900

MRoost 1600

Barley: Franse

Esterelle

Water Water

CTAB 1 +++ S ++ S D - - D - - - +++ S - - 210306 230306 2 +++ S ++ S D - - D - - - +++ S - - Dneasy 1 +++ S + S - D - - - D - - - ++ S - - 220306 220306 2 +++ S + S - D - - - D - - - ++ S - - Meyer 1 ++ S,D + S,D S,D D - HD - - - S,D - - 270306 270306 2 ++ S,D + S,D S,D D - HD - - - D - - Table 9 Scoring DNA Series 3: 4 types of hop products

Method

Hop flowers 100 mg

Hop flowers 20 mg

Hop pellets T90:

100 mg

Hop pellets T90:

20 mg

Isomerised hop

pellets: 100 mg

Isomerised hop

pellets: 20 mg

Hop extract: 100 mg

Water

CTAB 1 + S + S - + S + S + S - - 290306 2 + S + S - - + S - - - Dneasy 1 + S +/- S ++ S + S + S + S - - 300306 2 + S +/- S ++ S + S + S + S - - Meyer 1 +/- S - S - - ++ S,D ++ S - - 300306

2 +/- S - S - - ++ S,D ++ S - -

25

2. PCR RESULTS

4 different PCRs were done (see section 2.3):

1. Using PLANT 1 and PLANT 2 primers, that amplify an amplicon of 500-600 base pairs.

This PCR was done on all samples in all 3 series’.

2. Using rbcL-CRAw-Forward and rbcL-Reverse primers, that amplify an amplicon of 106

base pairs. This PCR was done on all samples in all 3 series’.

3. Using HOR 1B and HOR 2 primers, that amplify an amplicon of 197 base pairs. This PCR

was done on the samples in Series 2, as it is barley specific.

4. Using HOR 1B and HOR 2D primers, that amplify an amplicon of 645 base pairs on the

same gene as HOR 1B and 2. This PCR was also only done on the samples in Series 2.

After PCR, agarose gel electrophoresis was done. The bands produced were interpreted

according to their presence, size and intensity and the findings are summarised in Tables 10 to

12 along with the results from the extraction (Tables 7 to 9). All samples are in duplicate as 1

and 2 and the non-template controls are designated “NTC”.

Legend for tables “Scoring PCR results”

++ Definite positive amplification product

+ Positive amplification product

+/- Doubtful amplification product

- Negative/ no amplification product

Table 10 Scoring PCR results Series 1: Tobacco leaf and Pilot Experiment

Method PCR Tobacco

leaf Barley Malt Hop pellets Water NTC

CTAB 1 + +++ S +++ S - - CTAB 2 + +++ S +++ S - -

PLANT 1- PLANT 2 1 ++ ++ ++ - +/- -

PLANT 1 -PLANT 2 2 ++ ++ ++ - -

DNeasy 1 +++ + +++ S ++ S - DNeasy 2 +++ + +++ S ++ S -

PLANT 1- PLANT 2 1 ++ ++ ++ ++ +

PLANT 1- PLANT 2 2 ++ ++ ++ ++ +/-

Meyer 1 +++ + D ++ S,D - - Meyer 2 + + D ++ S,D - -

PLANT 1- PLANT 2 1 ++ ++ - +/- + -

PLANT 1- PLANT 2 2 ++ ++ - - +

26

Table 11 Scoring PCR results Series 2: 9 malt types and 1 barley variety

80°C 90°C 107°C 132°C 145°C 180°C 215°C 225°C 250°C

Method PCR

Pilsmout Ambermout Cara 20 Cara 120

Cara 300

MRoost 50

MRoost 450

MRoost 900

MRoost 1600

Barley: Franse

Esterelle

Water Water NTC NTC

CTAB 1 +++ S ++ S D - - D - - - +++ S - - CTAB 2 +++ S ++ S D - - D - - - +++ S - -

PLANT 1- PLANT 2 1 ++ ++ + + - - - - - ++ - +/- - ++

PLANT 1- PLANT 2 2 ++ ++ + - - + - - - ++ - -

RBCL-F- RBCL-R 1 + +/- +/- - - - - - - - - +/- +/-

RBCL-F- RBCL-R 2 +/- + + - - - - - - - - +/-

HOR 1B-HOR 2 1 ++ ++ ++ + - + - - - ++ - - -

HOR 1B-HOR 2 2 ++ ++ ++ +/- - ++ - - - ++ - +

HOR 1B-HOR 2D 1 ++ ++ + - - - - - - ++ - - -

HOR 1B-HOR 2D 2 ++ ++ - +/- - +/- - - - ++ - -

DNeasy 1 +++ S + S - D - - - D - - - ++ S - - DNeasy 2 +++ S + S - D - - - D - - - ++ S - -

PLANT 1- PLANT 2 1 ++ ++ + + + + +/- - - ++ - - -

PLANT 1- PLANT 2 2 ++ ++ + + + +/- - - - ++ - +/-

RBCL-F- RBCL-R 1 ++ ++ ++ + + ++ - - - ++ + + + +

RBCL-F- RBCL-R 2 ++ ++ ++ + + ++ - - - ++ + +

HOR 1B-HOR 2 1 ++ ++ ++ - - + - - - ++ - -

HOR 1B-HOR 2 2 ++ ++ ++ +/- - + - - - ++ - -

27

80°C 90°C 107°C 132°C 145°C 180°C 215°C 225°C 250°C

Method PCR


Cara 300

MRoost 50

MRoost 450

MRoost 900

MRoost 1600

Barley: Franse

Esterelle

Water Water NTC NTC

HOR 1B-HOR 2D 1 ++ ++ - - - - - - - ++ - -

HOR 1B-HOR 2D 2 ++ ++ - - - - - - - ++ - -

Meyer 1 ++ S,D + S,D S,D D - HD - - - S,D - - Meyer 2 ++ S,D + S,D S,D D - HD - - - D - -

PLANT 1- PLANT 2 1 - ? - - +/- +/- +/- +/- - - + +/- + - -

PLANT 1- PLANT 2 2 - - - +/- +/- +/- - - - +/- +/- +

RBCL-F- RBCL-R 1 - - - +/- +/- + +/- - - + + + +

RBCL-F- RBCL-R 2 - - - +/- + + - - - - + +

HOR 1B-HOR 2 1 - - +/- + - + - - - ++ + - -

HOR 1B-HOR 2 2 - - + +/- - + - - - ++ - -

HOR 1B-HOR 2D 1 - - - +/- - +/- - - - ++ - - -

HOR 1B-HOR 2D 2 - - - - - - - - - ++ - -

21 210306 220306 270306

230306 220306 270306

28

Table 12 Scoring PCR results Series 3: 4 types of hop products

Method PCR

Hop flowers 100 mg

Hop flowers 20 mg

Hop pellets T90:

100 mg

Hop pellets T90:

20 mg

Isomerised hop

pellets: 100 mg

Isomerised hop

pellets: 20 mg

Hop extract: 100 mg

Water NTC

CTAB 1 + S + S - + S + S + S - - 290306CTAB 2 + S + S - - + S - - -

PLANT 1- PLANT 2 1 ++ ++ + ++ ++ ++ +/- +/- -

PLANT 1- PLANT 2 2 ++ ++ + ++ ++ ++ +/- +/-

RBCL-F- RBCL-R 1 - +/- - ++ - - +/- + +

RBCL-F- RBCL-R 2 - + - ++ +/- + + +

DNeasy 1 + S +/- S ++ S + S + S + S - - 300306DNeasy 2 + S +/- S ++ S + S + S + S - -

PLANT 1- PLANT 2 1 ++ ++ ++ ++ ++ ++ - + -

PLANT 1- PLANT 2 2 ++ ++ ++ ++ ++ ++ +/- +/-

RBCL-F- RBCL-R 1 - ++ +/- ++ - + + + +

RBCL-F- RBCL-R 2 - ++ +/- + +/- + + +

Meyer 1 +/- S - S - - ++ S,D ++ S - - 300306Meyer 2 +/- S - S - - ++ S,D ++ S - -

PLANT 1- PLANT 2 1 - - +/- + - - + + -

PLANT 1- PLANT 2 2 - + - + - - +/- +/- -

RBCL-F- RBCL-R 1 - - - + - - + + +

RBCL-F- RBCL-R 2 - - - + - - + + +

29

3. SPECTROPHOTOMETRY RESULTS

Samples were analysed with a spectrophotometer that measured absorbance at 230nm,

260nm, 280nm and 320nm, as well as calculating the ratios A260/A280 and A260/A230 and the

concentration of DNA in µg/ml of the samples. A selection of samples from all 3 series were

analysed. The selection was done according to the results on agarose gel after extraction:

samples with reasonably good quality DNA were chosen. The ratio A260/A280 are summarised

in Tables 13 to 15 together with the DNA results on agarose gel. Table 16 displays all the

spectrophotometric results of all the samples analysed. The duplicates are shown as 1 and 2.

Legend for tables “Scoring of DNA”

+++ High concentration band

++ Medium concentration band

+ Low concentration band

- Negative sample

S Smeared

D Degraded

HD Highly degraded

For A260 below 0,1

For A260 above 1,0

Table 13 Scoring spectrophotometry results Series 1: Tobacco leaf and Pilot Experiment

Method

Tobacco leaf

Barley Malt Hop pellets

Water

CTAB 1 + +++ S +++ S - -

2 + +++ S +++ S - -

Ratio A260/A280

1 1,71 1,80 1,84 1,47

Ratio A260/A280

2 1,78 1,80 1,83 1,50

DNeasy 1 +++ + +++ S ++ S -

2 +++ + +++ S ++ S -

Ratio A260/A280

1 1,73 1,64 1,66 1,52

Ratio A260/A280

2 1,75 1,67 1,65 1,62

Meyer 1 +++ + D ++ S,D - -

2 + + D ++ S,D - -

Ratio A260/A280

1 1,77 2,17 1,94 -1,26 1,17

Ratio A260/A280

2 1,78 2,28 1,84 1,00

30

Table 14 Scoring spectrophotometry results Series 2: 9 types of malt and 1 barley variety

80°C 90°C 107°C 132°C 145°C 180°C 215°C 225°C 250°C

Method


Cara 300

MRoost 50 MRoost 450

MRoost 900

MRoost 1600

Barley: Franse

Esterelle

Water Water

CTAB 1 +++ S ++ S D - - D - - - +++ S - - 2 +++ S ++ S D - - D - - - +++ S - -

Ratio A260/A280 1 1,80 1,22 1,71 1,29 1,55 1,57 1,19

Ratio A260/A280 2 1,80 1,55 1,47 1,28 1,53 1,63

DNeasy 1 +++ S + S - D - - - D - - - ++ S - - 2 +++ S + S - D - - - D - - - ++ S - -

Ratio A260/A280 1 1,58 1,40 1,39 1,19 1,34 1,38 1,29

Ratio A260/A280 2 1,57 1,37 1,31 1,20 1,32 1,44

Meyer 1 ++ S,D + S,D S,D D - HD - - - S,D - - 2 ++ S,D + S,D S,D D - HD - - - D - -

Ratio A260/A280 1 1,90 1,62 1,75 1,50 1,80 1,73 1,14

Ratio A260/A280 2 1,89 1,62 1,80 1,54 1,85 1,72

21 210306 220306 270306

230306 220306 270306

31

Table 15 Scoring spectrophotometry results Series 3: 4 types of hop products

Method

Hop flowers 100 mg

Hop flowers 20 mg

Hop pellets T90:

100 mg

Hop pellets T90:

20 mg

Isomerised hop

pellets: 100 mg

Isomerised hop

pellets: 20 mg

Hop extract: 100 mg

Water

CTAB 1 + S + S - + S + S + S - - 290306,00 2 + S + S - - + S - - -

Ratio A260/A280 1 1,49 1,42 1,43 1,64 1,52 1,21

Ratio A260/A280 2 1,48 1,31 1,21 1,59 1,21

DNeasy 1 + S +/- S ++ S + S + S + S - - 300306,00 2 + S +/- S ++ S + S + S + S - -

Ratio A260/A280 1 1,50 1,42 1,63 1,55 1,42 1,35 1,11 1,26

Ratio A260/A280 2 1,50 1,41 1,64 1,53 1,43 1,36 1,19

Meyer 1 +/- S - S - - ++ S,D ++ S - - 300306,00 2 +/- S - S - - ++ S,D ++ S - -

Ratio A260/A280 1 1,39 1,27 1,21 1,68 1,61 1,15

Ratio A260/A280 2 1,24 1,21 1,25 1,63 1,52

32

Table 16 Spectrophotometric data

Sample Method Conc in µg/ml A230 A260 A280 A320

Ratio A260/A280

Ratio A260/A230

Tobacco 1 CTAB -9 -0,066 -0,017 -0,028 -0,014 0,61 0,26

Tobacco 2 CTAB 4 -0,049 0,007 -0,016 -0,014 -0,44 -0,14

Tobacco 1 DNeasy 27 -0,020 0,053 0,012 -0,014 4,42 -2,65

Tobacco 2 DNeasy 33 -0,011 0,065 0,018 -0,014 3,61 -5,91

Tobacco 1 Meyer 88 0,041 0,176 0,080 -0,008 2,20 4,29

Tobacco 2 Meyer 21 -0,050 0,041 0,004 -0,007 10,25 -0,82

Hop 1 CTAB 11 0,077 0,022 0,015 0,004 1,47 0,29

Hop 2 CTAB 2 0,008 0,003 0,002 -0,002 1,50 0,38

Malt 1 CTAB 128 0,122 0,256 0,139 -0,010 1,84 2,10

Malt 2 CTAB 139 0,126 0,278 0,152 0,000 1,83 2,21

Barley 1 CTAB 165 0,171 0,329 0,183 0,003 1,80 1,92

Barley 2 CTAB 109 0,106 0,218 0,121 0,002 1,80 2,06

Hop 1 DNeasy 69 0,106 0,138 0,091 0,012 1,52 1,30

Hop 2 DNeasy 83 0,105 0,165 0,102 0,007 1,62 1,57

Malt 1 DNeasy 150 0,194 0,299 0,180 0,026 1,66 1,54

Malt 2 DNeasy 132 0,165 0,263 0,159 0,025 1,65 1,59

Barley 1 DNeasy 36 0,051 0,072 0,044 0,006 1,64 1,41

Barley 2 DNeasy 38 0,054 0,075 0,045 0,005 1,67 1,39

Hop 1 Meyer -15 0,040 -0,029 0,023 0,003 -1,26 -0,73

Hop 2 Meyer 16 0,054 0,031 0,031 0,011 1,00 0,57

Malt 1 Meyer 394 0,424 0,788 0,407 0,029 1,94 1,86

Malt 2 Meyer 337 0,392 0,674 0,366 0,039 1,84 1,72

Barley 1 Meyer 131 0,127 0,262 0,121 0,013 2,17 2,06

Barley 2 Meyer 103 0,094 0,205 0,090 0,008 2,28 2,18

Water 1 Meyer -28 -0,059 -0,055 -0,047 -0,012 1,17 0,93

Pilsmout 1 CTAB 109 0,048 0,218 0,101 -0,014 2,16 4,54

Pilsmout 2 CTAB 84 0,034 0,167 0,073 -0,014 2,29 4,91

Ambermout 1 CTAB 164 0,445 0,328 0,267 0,138 1,23 0,74

Ambermout 2 CTAB 41 0,018 0,082 0,043 -0,005 1,91 4,56

Cara 20 1 CTAB 34 -0,005 0,067 0,021 -0,010 3,19 -13,40

Cara 20 2 CTAB 62 0,084 0,123 0,073 0,007 1,68 1,46

Cara 120 1 CTAB 13 0,051 0,026 0,016 0,004 1,63 0,51

Cara 120 2 CTAB 10 0,032 0,019 0,011 0,002 1,73 0,59

MRoost 50 1 CTAB 66 0,066 0,132 0,071 -0,003 1,86 2,00

MRoost 50 2 CTAB 65 0,063 0,129 0,071 -0,003 1,82 2,05

F. Esterelle 1 CTAB 89 0,103 0,178 0,099 0,005 1,80 1,73

F. Esterelle 2 CTAB 121 0,121 0,241 0,132 0,007 1,83 1,99

Water 1 CTAB 0 0,006 0,000 0,001 0,001 0,00 0,00

Pilsmout 1 DNeasy 145 0,165 0,289 0,169 0,010 1,71 1,75

Pilsmout 2 DNeasy 128 0,136 0,255 0,148 0,012 1,72 1,88

Ambermout 1 DNeasy 47 0,066 0,093 0,057 0,005 1,63 1,41

Ambermout 2 DNeasy 35 0,049 0,070 0,044 0,007 1,59 1,43

Cara 20 1 DNeasy 55 0,090 0,109 0,071 0,017 1,54 1,21

Cara 20 2 DNeasy 57 0,102 0,113 0,081 0,021 1,40 1,11

Cara 120 1 DNeasy 30 0,077 0,060 0,051 0,022 1,18 0,78

Cara 120 2 DNeasy 24 0,061 0,047 0,039 0,017 1,21 0,77

MRoost 50 1 DNeasy 65 0,112 0,130 0,092 0,044 1,41 1,16

MRoost 50 2 DNeasy 38 0,070 0,075 0,052 0,014 1,44 1,07

F. Esterelle 1 DNeasy 72 0,137 0,143 0,096 0,020 1,49 1,04

33

F. Esterelle 2 DNeasy 76 0,104 0,151 0,095 0,018 1,59 1,45

water 1 DNeasy 18 0,069 0,035 0,024 0,005 1,46 0,51

Pilsmout 1 Meyer 689 0,750 1,378 0,705 0,057 1,95 1,84

Pilsmout 2 Meyer 606 0,655 1,212 0,619 0,045 1,96 1,85

Ambermout 1 Meyer 257 0,369 0,513 0,300 0,062 1,71 1,39

Ambermout 2 Meyer 271 0,403 0,541 0,317 0,077 1,71 1,34

Cara 20 1 Meyer 346 0,435 0,692 0,378 0,067 1,83 1,59

Cara 20 2 Meyer 444 0,528 0,888 0,473 0,070 1,88 1,68

Cara 120 1 Meyer 150 0,260 0,299 0,188 0,063 1,59 1,15

Cara 120 2 Meyer 222 0,390 0,443 0,275 0,088 1,61 1,14

MRoost 50 1 Meyer 553 0,688 1,106 0,595 0,096 1,86 1,61

MRoost 50 2 Meyer 551 0,634 1,101 0,575 0,074 1,91 1,74

F. Esterelle 1 Meyer 182 0,202 0,364 0,192 0,018 1,90 1,80

F. Esterelle 2 Meyer 163 0,188 0,325 0,171 0,019 1,90 1,73

Water 1 Meyer 6 0,022 0,012 0,014 0,002 0,86 0,55 Hop flowers

100mg 1 CTAB 53 0,060 0,106 0,059 0,000 1,80 1,77 Hop flowers

100mg 2 CTAB 49 0,058 0,097 0,054 -0,001 1,80 1,67 Hop flowers

20mg 1 CTAB 23 0,016 0,045 0,023 -0,008 1,96 2,81 Hop flowers

20mg 2 CTAB 36 0,064 0,072 0,050 -0,001 1,44 1,13 Hop pellet

20mg 1 CTAB 37 0,044 0,074 0,042 -0,005 1,76 1,68 Hop pellet

20mg 2 CTAB 4 0,023 0,007 0,005 0,001 1,40 0,30 Isomerised hop pellets 100mg 1 CTAB 123 0,106 0,245 0,134 -0,005 1,83 2,31 Isomerised hop pellets 100mg 2 CTAB 98 0,100 0,196 0,109 0,000 1,80 1,96 Isomerised hop pellets 20mg 1 CTAB 41 0,075 0,082 0,041 0,001 2,00 1,09 Isomerised hop pellets 20mg 2 CTAB 13 0,046 0,026 0,021 0,008 1,24 0,57

Water 1 CTAB 4 0,023 0,008 0,006 0,002 1,33 0,35 Hop flowers

100mg 1 DNeasy 114 0,182 0,227 0,139 0,023 1,63 1,25 Hop flowers



20mg 2 DNeasy 41 0,069 0,082 0,049 0,003 1,67 1,19 Hop pellet 100mg 1 DNeasy 301 0,362 0,602 0,353 0,033 1,71 1,66

Hop pellet 100mg 2 DNeasy 324 0,403 0,648 0,380 0,040 1,71 1,61



Isomerised hop pellets 100mg 1 DNeasy 135 0,202 0,269 0,179 0,042 1,50 1,33 Isomerised hop pellets 100mg 2 DNeasy 99 0,177 0,198 0,129 0,034 1,53 1,12 Isomerised hop pellets 20mg 1 DNeasy 40 0,071 0,080 0,053 0,008 1,51 1,13 Isomerised hop pellets 20mg 2 DNeasy 35 0,053 0,069 0,044 0,003 1,57 1,30

Hop extract 1 DNeasy 17 0,056 0,034 0,035 0,007 0,97 0,61

Hop extract 2 DNeasy 7 0,028 0,013 0,011 0,037 1,18 0,46

Water 1 DNeasy 4 0,013 0,007 0,003 -0,001 2,33 0,54 Hop flowers

100mg 1 Meyer 51 0,106 0,102 0,067 0,018 1,52 0,96

34

Hop flowers

100mg 2

Meyer

57

0,116

0,113

0,073

0,016

1,55

0,97 Hop flowers

20mg 1 Meyer 19 0,054 0,038 0,028 0,005 1,36 0,70 Hop flowers

20mg 2 Meyer 19 0,052 0,037 0,026 0,006 1,42 0,71 Hop pellet 100mg 1 Meyer 6 0,039 0,012 0,014 0,003 0,86 0,31

Hop pellet 100mg 2 Meyer 11 0,050 0,022 0,015 0,003 1,47 0,44

Isomerised hop pellets 100mg 1 Meyer 266 0,364 0,532 0,302 0,034 1,76 1,46 Isomerised hop pellets 100mg 2 Meyer 330 0,475 0,660 0,388 0,053 1,70 1,39 Isomerised hop pellets 20mg 1 Meyer 163 0,211 0,325 0,186 0,020 1,75 1,54 Isomerised hop pellets 20mg 2 Meyer 111 0,156 0,222 0,133 0,012 1,67 1,42

Water 1 Meyer 3 0,015 0,005 0,007 0,000 0,71 0,33

7 µl AE-buffer +

63 µl H2O 36 0,098 0,072 0,060 0,014 1,20 0,74

Please note: The blank in this table is a 1/10 dilution of AE-buffer. The values obtained from this blank were subtracted from the samples’ measurements which were measured with HPLC H2O as a blank. Samples with A260 between 0.1 and 1.0 Samples with A260 above 1.0

Table 17 Summary

Mean St. dev

Method

ratio OD260/OD280

with OD260 0.1<x<1

ratio OD260/OD280

with OD260 0.1<x<1

Number of

samples included

CTAB 1.82 0.225 n=15 DNeasy 1.60 0.104 n=19 Meyer 1.82 0.215 n=19

DISCUSSION

The results we obtained were used to evaluate the DNA extraction methods using the intensity

and existence of the bands on agarose gel after DNA extraction, the intensity and existence of

bands on agarose gel after PCR and the values from the spectrophotometric analysis. Intense

bands on agarose gel were taken to mean that there was a fair amount of DNA in the sample

or amplification product, while the occurrence of no band was taken to be a negative sample,

that is an unsuccessful extraction or amplification. First, the appearance of the bands on

agarose gel after DNA extraction will be discussed and, following that, the appearance of the

bands after PCR amplification and, lastly, the spectrophotometric results.

35

With the CTAB protocol, DNA of reasonable quality (as interpreted from the agarose gels)

was extracted from Pilsmout, Ambermout and Franse Esterelle. Cara 20 and MRoost 50 gave

rise to degraded DNA with no bands with high molecular weight DNA, while the other 5 malt

types did not yield any DNA bands that could be seen on agarose gel. This is probably

because these 5 malt types have been treated with the highest temperatures: heat is known to

fragmentise DNA [8]. Interestingly, MRoost 50 is treated with a higher temperature than both

Cara 120 and 300 but in contrast to them does show a yield of DNA from the extraction.

However, during MRoost production, this high temperature was applied to Pilsmout as

starting material, which is dry material, whereas for the Cara malts the temperature was

applied to green malt. The MRoost 50 is also derived from a different barley variety

compared to Cara 120, 300 and the other MRoost varieties and may withstand heat better. See

Table 1 for details.

It is also the DNA from Pilsmout, Ambermout, Cara 20, MRoost 50 and Franse Esterelle

extracted with the CTAB protocol that show positive amplification during PCR. Particularily,

amplification with the primer pairs PLANT 1 and 2, HOR 1B and 2 and HOR 1B and 2D

show good results, while the primer pair rbcL-Forward and Reverse does not show the same

clear results. This is despite the fact that the amplicon length of both PLANT 1 and 2 and the

HOR primer pairs is larger than that of the rbcL primer pair, which rules out amplification

difficulty because of the amplicon size. A likely explanation is that this is due to that the rbcL

PCR requires 3.75µl of the DNA sample whereas the other PCR protocols require only 1µl of

the DNA sample. If the DNA sample is contaminated with PCR-inhibitors, a larger amount of

sample will also include a correspondingly larger amount of inhibitors that will be included in

the PCR-mix, which could explain the results. From the bands present on agarose gel after

PCR we can also see that, using the HOR primer pairs, it is easier to amplify the 197bp

fragment than the 645bp fragment. Only bands from Pilsmout, Ambermout and Franse

Esterelle are present, showing clear amplification of the larger amplicon size for DNA of

these 3 samples. These 3 have been treated the least (the barley not at all) which could explain

the results as the DNA is more conserved in these sample types. The degraded DNA isolated

from Cara 20, 120 and MRoost 50 can be amplified with the HOR primer pair producing the

smaller HOR amplicon (197bp) but not with the HOR primers designed to produce the larger

(645bp), indicating that their DNA is in smaller fragments than about 600bp. As the CTAB

protocol is the only method that does not involve a commercial kit, the results are adequate,

considering that the extraction conditions are less controlled. The DNA integrity and amount

extracted is slightly better than that for DNeasy, as can be seen from the intensity of the high

36

molecular weight DNA bands on agarose gel after extraction. However, apart from the HOR

primer pairs, the PCR results are of a lower standard than that of DNeasy and the number of

DNA smples that can be amplified is lower, which can possibly be explained by the presence

of more PCR-inhibitors.

The DNeasy protocol allowed us to obtain DNA of comparable integrity after extraction to

CTAB DNA with visible bands on agarose gel after extraction for Pilsmout, Ambermout,

Cara 20, MRoost 50 and Franse Esterelle. However, the intensity of the bands is less than for

CTAB, indicating that the concentration of the DNA extracted by DNeasy is lower. This can

be seen on the agarose gels when comparing the bands’ intensities with each other and using

the λ-DNA bands as a reference. Despite this, the results after PCR using template DNA

obtained from DNeasy are often considerably better than using CTAB DNA, with more

definite results and amplification visible on gel with the PLANT and rbcL primer pairs.

Infact, it is only the MRoost 450, 900 and 1600 that show no amplification with PLANT and

rbcL, while both Cara 120 and 300 (that do not present visible bands on agarose gel after

DNA extraction) show amplification results for both PCRs, as can be seen by the presence of

bands on agarose gel after PCR amplification. This might be explained by the extraction

method’s ability to remove PCR contaminants therefore giving clearer results after

amplification, even with small amounts of template DNA.

The PCR results, as seen by the appearance and intensity of the bands on agarose gel, from

both HOR primer pairs are similar to those of the CTAB protocol, with the same types of malt

and the barley variety showing positive amplification for the smaller amplicon size (197bp).

Similarily, only the Pilsmout, Ambermout and Franse Esterelle show clear bands on agarose

gel after amplification of the larger HOR amplicon (645bp), again suggesting that these 3

samples have the most conserved DNA as a result of the relatively low temperature

treatments. Any physical or chemical treatment of food samples, such as heat, pH or shear

forces results in a decrease in the average DNA fragment size due to random cleavage [19].

The results with the DNeasy protocol have been the most consistent. Especially the PCRs

have been successful for most of the sample types.

The Meyer protocol gives the poorest results of the 3 methods with poorer intensity of the

bands visible on agarose gel after DNA extraction and shows considerable degradation and

smearing. Visible bands, though smeared, can be seen with the same malt types, Pilsmout,

Ambermout, Cara 20 and MRoost 50, and with Franse Esterelle, as with the DNA extracted

37

with the CTAB and DNeasy protocols. The degradation and smearing might be explained by

the powerful lysing buffer and the long incubation time during extraction. DNA samples

extracted with this protocol also show the poorest results after PCR with few visible positive

results of amplification with both PLANT 1 and 2 and rbcL-Forward and Reverse. This can

be seen by examining the agarose gels. There are hardly any bands present and the ones that

can be seen are faint. Interestingly, the only visible amplification results on agarose gel (apart

from Franse Esterelle) are found with Cara 120, Cara 300, MRoost 50 and MRoost 450,

which show either no DNA bands or degraded DNA on agarose gel after DNA extraction.

Contamination could be an explanation but it is not satisfactory since there are clear negative

samples after amplification as well.

Again with the HOR primer pairs there is amplification visible on agarose gel for Cara 20,

120, MRoost 50 and Franse Esterelle. Apart from the barley, the bands are faint, indicating

that the amplification is not entirely successful and that there is little useful DNA for

amplifiaction or that PCR-inhibitors are present. Amplification of the larger HOR amplicon is

almost entirely unsuccessful. Franse Esterelle is the only sample to give clear bands for the

645bp amplicon. There are faint bands for Cara 120 and MRoost 50 but they are far from

definite positives. The poor results obtained with DNA extrated with Meyer, compared to

CTAB DNA and DNeasy DNA, indicate that there is something about the extraction method

that affects the DNA quality. Most likely is that the powerful lysis agent and the long

incubation are too powerful for the sample types used in this project. Besides that, the DNA

extracted from Pilsmout seems to be of sufficient integrity and concentration on inspection of

the agarose gel, but no PCR amplification is successful, which suggests the presence of PCR-

inhibitors. However, since the Meyer extraction protocol apparently yields results for malt

types that do not yield results with the other DNA extraction methods, further research is

required.

None of the 3 protocols are as effective at extracting DNA from hop products as they are with

malt and barley. Generally, positive results seen after DNA extraction on agarose gel can be

seen for hop flowers, powdered hop pellets and isomerised hop pellets while the hop extract

does not show any visible bands. Because the hop absorbed a lot of fluid during extraction

and the results on agarose gel after extraction from the pilot experiment gave relatively poor

intensity bands with 100mg hop samples, we decided to extract DNA from both 100mg and

20mg of each type of hop product (except hop extract) This was to see whether a lower

amount of starting material might give better results. On the whole, this could not be verified

38

after DNA extraction as the bands that could be seen were similar for both amounts of starting

material. All bands after DNA extraction were smeared and none of the bands displayed any

considerable intensity. DNeasy was the only method that showed visible bands on agarose gel

with all 3 types of hop products while the hop pellets gave poor results with CTAB and

Meyer on gel. However, the Meyer protocol gave the best results with the most intense bands

with isomerised hop pellets although the DNA was degraded and smeared but the poorest

results with hop flowers with no visible bands present on agarose gel.

The PLANT 1 and 2 primer pair gave positive amplification, as could be seen by the

presence of bands with the correct size on agarose gel, with CTAB and DNeasy DNA from

hop flowers, hop pellets and isomerised hop pellets, despite some apparently negative samples

(no bands could be seen on agarose gel) after extraction with CTAB. The results with the

Meyer protocol are not as clear, with only the 20mg hop pellets DNA giving clear bands

(although faint bands could be seen with other samples), which is interesting as no DNA from

20mg hop pellets could be seen after DNA extraction on agarose gel. Also the hop extract and

water controls display bands after amplification with the PLANT primer pair. Conversely, the

DNA extracted with the Meyer protocol from isomerised hop does not show any

amplification with the PLANT PCR as no bands are visible on gel. The results are similar for

the Meyer DNA samples with the rbcL primer pair, with faint bands visible for 20mg hop

pellet DNA, hop extract and the water controls. The results from CTAB and DNeasy DNA

with the rbcL primer pair show differences to the results that can be seen on gel with the

PLANT primer pair. It can now be seen that the 20mg samples give better results with more

intense bands with this primer pair while the 100mg samples give negative results or doubtful

bands on agarose gel. This could be because the sample matrix may contain PCR-inhibitors

and the larger amount of sample would have more inhibitors compared to the smaller amount.

The inhibitors might be more easily extracted compared to the DNA, resulting in that the

extracted DNA from the larger sample volume contains a higher concentration of inhibitors.

For all 3 protocols, hop extract shows positive results, as can be seen by the faint bands on

gel, with both primer pairs. However, since the water controls also give very similar results,

this could be due to contamination during extraction.

Spectrophotometric analysis was done on a selection of samples that had displayed visible

bands (positive results) on agarose gel after extraction. With the criterium that pure DNA

samples should have a A260/A280 ratio above 1.8, about a ⅓ of the 101 samples can be said to

consist of pure DNA. Contamination with proteins lowers the ratio and this can be seen in

39

many cases, indicating that the extraction methods are not entirely successful at removing all

protein contamination. However, using cut-off values of A260 between 0.1 and 1.0 (between

which the relationship absorbance-concentration is linear), the mean ratio of A260/A280 of the 3

methods shows that both the CTAB method and the Meyer method have ratios above 1.8

which indicates high purity of the DNA extracted by these methods. The DNeasy, however,

does not (1.60), indicating that this method allows more protein contamination of the samples

which is interesting as the manufacturer claims it should give pure DNA. But, according to

PCR results on agarose gel, DNeasy-extracted DNA has the best quality for PCR

amplification, which is contradictory to the spectrophotometric results. The results could, at

least in part, be explained by that the possible PCR-inhibitors present in DNA samples

prepared with the CTAB or Meyer protocols have no influence on the A260/A280 ratio. Other

contradictions can be seen between the spectrophotometric and PCR results for the DNA

samples. For example, the Pilsmout and Ambermout DNA samples extracted with the CTAB

protocol have very different A260/A280 ratios but behave very similar in PCR amplification. In

another example, the A260/A280 ratio with DNeasy-extracted Cara 20 DNA has a very low

ratio value which would suggest a impure DNA, but PCR amplification (especially with the

HOR primers) works well. As a result, the conclusion can be drawn is that there is no obvious

relationship between the A260/A280 ratio and the “template quality” for PCR.

Only 53 of the 101 samples measured have an A260 that falls between the cut-off values.

The concentration of DNA in the samples varies a lot, from just a few µg/ml to over

600µg/ml. However, at this stage of the project we are not primarily concerned with DNA

quantity.

I found that one of the main advantages of the DNeasy and Meyer protocols was the amount

of time they took, compared to the CTAB protocol which took a whole day. The long

incubation time in Meyer is a disadvantage but it also means that it can be combined, time-

wise, with the DNeasy protocol or other laboratory work and so in the end is efficient. All 3

protocols require a lot of disposable material e.g. eppendorf tubes and pipette tips. Based on

the results and practical concerns, I am most in favour of using the DNeasy protocol for DNA

extraction of barley, malt and hop products. Terry et al reported various advantages and

disadvantages of each of the 3 methods: the CTAB protocol has good usage and results with

phenol/chloroform or chloroform followed by isopropanol precipitation and is effective for a

wide range of matrices. CTAB complexes out polysaccharides and proteins which reduces

PCR-inhibition but a disadvantage is that it has a 260nm effect during spectrophotometric

40

quantitation. With the DNeasy protocol, only a limited amount of sample material can be used

successfully and it does not yield good results with highly processed samples. It is non-toxic

but also expensive and gives a possible salt inhibitor of PCR. The use of SDS in the Meyer

method improves lysis but also gives a possible protein inhibitor of PCR [7].

Only one barley variety was included in this study, but the malt types used originate from

different barley varieties. For full comparison between the results from the malt and the

results from the original untreated barley, all barley varieties used for the malting process

would need to be analysed. Further, I recommend using a more powerful grinding machine to

fully grind the barley seeds to a powdered form to ensure that all plant material is included in

the extraction.

It is possible from the results we have to date to conclude that temperature does have an effect

on the quality of DNA extracted from malt. The general trend is that the higher the

temperature treatment the samples are exposed to, the poorer the integrity of the extracted

DNA. However, the MRoost varieties of malt are a double treated malt, made by retreating

Pilsmout malt. Therefore, to properly evaluate the effects of temperature on the quality of

extracted DNA, we would need to use malt types that have not undergone the double

treatment but have instead only been subjected to one temperature treatment during the

malting process, as described on page 5, as well. Therefore, more malt types would need to be

analysed to draw any define conclusions as to whether only a high temperature or the double

treatment has a more pronounced effect on DNA quality.

We analysed only 9 types of malt with 3 extraction methods. There are many more types of

malt available; a survey among breweries in the area would reveal the most commonly used

types which could then be analysed. We have not been able to extract good quality DNA from

Cara 300, MRoost 450, 900 and 1600 and perhaps another extraction method could be

successfully used with these malt types. Other extraction methods that can be tested are the

Invisisorb Spin plant genomic purification kit from Westburg, the Wizard method from

Promega, the ROSE method, Alkali method and many others. Generally the commercial kits

have low recovery rates but good DNA quality while other methods give a higher recovery

rate but poor DNA quality [20]. In our case, the DNA quality is of greater importance and this

favours the use of commercial kits. The term “DNA quality” is defined as the degree of

degradation of DNA and by the presence or absence of potential inhibitors of the PCR [19].

41

The DNA extraction protocols must be developed on a case-by-case basis and adapted to fit

the sample matrix, material availability and the intended use of the extracted DNA [21]. In

general, potential extraction methods must be validated using several factors: the yield,

quality and integrity of the DNA extracted; the type of sample matrix that can be successfully

extracted using the method; the presence of potential inhibitors of downstream processes e.g.

PCR amplification; and the repeatability of the technique. There also needs to be a balance

between the ease of use, cost, and generic application of a particular technique versus the

quality and quantity of DNA obtained and the type of matrix [7].

To successfully obtain DNA from hop extract, I suggest trying the protocol as described in

reference 5. It describes how DNA is extracted from olive oil using a CTAB protocol with

high detergent concentration, cold centrifugation and freeze/thaw cycles with a larger amount

of starting material. The olive DNA obtained with this method is amplifiable with a PCR and

can be used for AFLP analysis [5]. DNA has also been extracted successfully from soybean

oil using the Wizard method [22] as well as from rapeseed oil using mini-concentrators and a

nucleus digestion buffer [23]. These methods could also be successful with hop extract

because of its oily character.

We have had problems with the negative water controls during extraction showing positive

results after PCR. This could be due to contamination during extraction since the water

samples where always last in every extraction sequence which greatly adds to the

contamination risk. The only remedy for this is to redo the extraction protocols, maybe with

fewer samples per extraction “round” and more care not to have more than one sample tube

open at any one time. Because the water controls sometimes show positive results after PCR,

the doubtful positive bands after agarose gel electrophoresis can not be definitely determined

either way, since they could also be contaminated negatives or simply contain a low amount

of DNA or amplification product. A limitation of PCR-based methods of identification is

false-positive results due to accidental contamination of the sample or reagents e.g. cross-

contamination between samples. This can be reduced in several ways e.g. using separate sets

of supplies and pipettes, UV-light and the physical separation of sample preparation, pre-PCR

and post-PCR [19]. All these measures were taken.

Other measures that can be considered during extraction to increase the quality and yield of

DNA are the use of α-amylase in the lysis step to aid the breakdown of starch [7], and the

42

clean-up of extracted DNA as an extra step during extraction using e.g. a commercial kit like

Wizard DNA Clean-up System by Promega, which has a positive effect on the amplification

of DNA. During PCR the use of a hot-start Taq polymerase can be used to avoid the non-

specific amplification products that can form using normal Taq polymerase [21].

The next step towards variety identification is DNA fingerprinting of the extracted DNA. This

is the purpose of DNA extraction. DNA fingerprinting methods will need to be researched

and tested before the samples can be fingerprinted and the malt types satisfactorily traced to

the barley varieties they are derived from and the hops varieties confirmed. It is important that

the methods that are decided on can be used to verify that a variety indeed comes from the

source the producer has stated. Traditionally, hops cultivar identification has been verified by

chemical analysis or by morphological characteristics. However, both these traits are

influenced by the environmental conditions during cone development. Since hop cultivars are

clonally propagated (all individuals in a cultivar are therefore genetically identical), DNA

fingerprinting using a molecular marker approach is an ideal way to distinguish among hop

cultivars. A technique for analysing hop DNA would allow a brewer to verify a hop sample’s

purity before the brewing process [4].

Fingerprinting methods that can be considered are:

Restriction Fragment Length Polymorphism (RFLP): restriction fragments from a given

chromosomal locus often vary in length in different individuals of the same species. They

have their origin in base sequence changes or DNA rearrangements and are naturally

occurring, simply inherited, Mendelian characters. This method requires large quantities of

high quality DNA and is not a PCR-based technique.

Hybridisation-based mini and microsatellite fingerprinting (VNTR): is similar to RFLP.

Hybridisation is carried out with oligonucleotide probes for mini/microsatellite sequences.

Several genetic loci are assayed simultaneously which gives multilocus fingerprinting. Each

locus is characterised by more or less regular arrays of tandemly repeated DNA motifs that

occur in different numbers at different loci.

Single Sequence Repeat (SSR) PCR assay: these are DNA sequences that consist of 2-5

nucleotide core units tandemly repeated. They are spread through the genome of eukaryotes.

They form the basis of a PCR-based, multi-allelic, co-dominant genetic marker system. Since

the flanking sequences are unique and conserved among genotypes of the same species,

primers to the flanking regions can be designed to define a sequence tagged microsatellite.

43

Random Amplified Polymorphic DNA (RAPD): this is a versatile method that generates

fingerprints of genomes using arbitrarily selected primers under conditions where primers will

initiate synthesis of DNA, even when the match with the template is imperfect. It is quick,

simple and efficient. DNA probes and sequence information for primer design is not needed.

It only requires a thermocycling machine and agarose gel electrophoresis apparatus both of

which are available to us. The results however, can be difficult to reproduce.

Amplified Fragment Length Polymorphism (AFLP): it involves restriction digestion and PCR

and is based on sequence polymorphisms at nucleotide level. Single nucleotide changes can

be detected. Deletions, insertions and rearrangements affecting the presence or size of the

restriction fragments will lead to AFLP length polymorphisms. The method is robust and

reproducible. In short, it involves the ligation of double-stranded adaptors to the ends of

restriction fragments. These serve as primer binding sites in the subsequent amplification

steps. Next, PCR amplification of the subsets using selective AFLP primers is done. Detection

of the AFLP fragments is by labelling of 1 of the 2 AFLP primers with radioactive isotopes or

fluorescence [24]. The AFLP technique is a recent innovation that is powerful and reliable. It

has several advantages: very little genomic DNA is needed, many polymorphisms can be

generated per reaction, prior knowledge of specific DNA sequences is not needed and the

technique has high reproducibility [4]. In one example, 31 barley varieties were identified

with only 8 AFLP primer combinations [25]. This method has been shown to be useful for

verifying hops identity and purity using DNA extracted from dried hop cones [4].

Single Nucleotide Polymorphisms (SNPs): this has until recently been restricted to the human

genome but is now also used in agriculture and horticulture. SNPs are single-base variations

in the genetic code and are the most prevalent class of genetic markers for linkage analysis.

They are bialellic, frequent and mutationally stable. Once the SNP locus is identified and

characterised they can be detected using PCR-based approaches among other methods [24].

It has been found that DNA extracted from dry whole malt is partially degraded and

unsuitable for fingerprinting. To overcome this problem, it is suggested that DNA is extracted

from coleoptiles taken from malted seeds before kilning which results in comparable quality

to that from fresh young barley leaves. The extraction yield is also found to be satisfactory.

However, this is not an option once the malt is purchased from the brewer. The poor identity

of RAPD patterns between a barley variety and the corresponding malt make these markers

unsuitable for malt characterisation. However, AFLP analysis based on template DNA

extracted from grain malt coleoptiles is an excellent tool for malt fingerprinting with high

44

repeatability and analysis speed [26]. It will be necessary for us to test the DNA fingerprinting

techniques chosen on the malt types we have, despite them being dried and kilned.

In conclusion, we have been able to extract DNA of good quality from barley and malt using

the CTAB and DNeasy protocols. The extracted DNA is suitable for PCR as an example of a

downstream application. The Meyer protocol is not as successful at extracting DNA of good

integrity. Hop products do not, on the whole, give DNA of the same good quality as malt and

barley. The DNeasy protocol is the most successful with hops. Temperature treatment of malt

as well as hop processing has an adverse effect on the DNA integrity. More research is

needed both at this step of the project and in the next stage.

ACKNOWLEDGEMENT

I would most sincerely like to thank Dr. Leen Van Houdt, the best supervisor a student could

hope for. Her kindness, patience and her skills as a teacher are truly appreciated. I would also

like to thank her for all her work with the scanning of all the gel pictures, the tables and for

going through this paper endless times. Additionally, I would like to thank all the staff of the

Campus Gildestraat, KaHo Sint-Lieven, for all their help and kindness to me. Finally, the

Erasmus Programme deserves a mention as well for the opportunity to do my final thesis in

another country, which has been a great experience.

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Documents

Evaluation of DNA Quality of Beer Ingredients Anna Ramberg131019/FULLTEXT01.pdfresponsible for making beer [3]. Ale yeast is usually used for fermentation at temperatures above 15°C