Extraction Methodologies

1Extraction Methodologies -Anthropology/ Biology 3138 Apryle Berube, November 17, 2010

Extraction Methodologies

Anthropology/ Biology 3138

Group 3

Presented to Dr. Matheson

Wednesday 9:30-12:30

Apryle Berube(0444381)

___________________________

Teaching Assistant: Franz Masini

__________________________

November 17, 2010

Lakehead University


Introduction

DNA extraction is need to study DNA such as in polymerase chain reaction(PCR) amplification.

The extraction of DNA is comprised of four basic steps which included collection of the cells,

lysing of the cells, removal of protein, and collection of DNA. [2] There are many ways to collect

cells for a sample. One way is to put out a hair with the root intact. Another way is to simply

wipe the inside of a cheek with a cotton swab or swish NaCl in one’s mouth. Basically, all that is

needed in this step is a collection of cells to be studied. There are many reagents used for DNA

extraction, two of which are studied in this lab. The Chelex extraction is a physical extraction

method and the guanadinium thiocyanate/ silica bead method is a chemical extraction. The first

step in DNA extraction is lysing if the cells. Lysing the cells requires a reagent or heat to break

open the cells and release the DNA. There are many ways to do this. In a physical extraction,

such as Chelex, the cells are lysed by heating them in the Chelex solution. This also dentaures

the DNA in a single strand.The guanadinium thiocyanate solution lyses the cells and digests

protein in the sample. [3] Next is the removal of protein. Chelex is a physical extraction and does

not involve purification steps in the extraction, so protein is not removed. For the guanadinium

thiocyanate solution, a wash buffer is added to remove the protein and other cellular debris,

therefore purifying the sample. Collection of DNA in both extraction methods results in the

DNA being suspended in the solution. Certain factors should be considered when selecting an

extracting agent. For example, Chelex is best for the extraction of DNA used in PCR since it

removes contaminating metal ions that catalyze the digestion of DNA.[2] Guanadinium

thiocyanate/ silica bead solution is useful in the extraction of DNA from tissue and hair samples.

[5]


To further purify samples, sodium acetate is usually added. The sodium ions help to neutralize

the negative charge on the phosphate groups of the DNA backbone thereby precipitating the

DNA. Cold ethanol is then used to precipitate the remaining DNA since it is not soluble in cold

ethanol. The supernatant is removed and what is left is a purified sample.

Once data for DNA sample have been read on a spectrophotometer, the ratio of DNA/protein

must be calculated as well as DNA concentration. Protein in a sample is measured at 280nm and

DNA is measured at 260nm, therefore the ratio of 260nm/280nm equals the amount of DNA in

relation to the amount of protein. This is useful in determining purity of a sample. The ideal ratio

for this is over 1.8 which would indicate approximately 50% protein and 50% DNA in the

sample. The concentration of DNA in a sample is calculated by the formula below:

[DNA]= 50uG/mL x 260nm optical density x dilution factor

To conserve DNA and fill the spectrophotometric cuvettes, the original sample must be diluted

with water. This affects the concentration of the sample hence the reason why the dilution factor

is important to the calculation of DNA concentration. The main reason for the calculation of

concentration of DNA for this experiment is ensure the proper amount of DNA added to

polymerase chain reaction amplification tubes.[1]

Objective

The purpose of this lab is to compare and understand different extraction methods, as well as

observing and quantifying the DNA obtained from a hair/root sample and a cheek cell sample

using spectrophotometric technique


Materials

Hair sample

Scissors

Tweezers

1.5mL eppendorf tubes

50mL conical tubes

500uL Guanidinium thiocyanate solution

-4M Guanadinium thiocyanate

-0.1M Tris-Hydrogenchloride

-0.02M Ethylenediaminetetraacetic acid

-1.3% triton X-100

~500uL Guanidinium thiocyanate solution + silica beads

Mini Vortex(Fisherbrand)

Ice Cooler

Mini Centrifuge(Fisherbrand)

200uL ethanol

500uL of wash buffer

-0.05M Sodium chloride

-50% ethanol

-1mM Ethylenediaminetetraacetic acid

-0.001M Tris-HCl

Sterilized H2O

100-1000 µL Pipette (Thermo Scientific)

20-200 µL Pipette (Thermo Scientific)

20-200 µL Pipette tips

100-1000 µL Pipette tips

Small plastic trash container


Cotton tip swab

500mL Chelex solution

Heat block(Isotemp)

Benchtop centrifuge(Serval Legend Micro)

Thermoscientific Genesys spectrophotometer

1.5mL cuvettes

22.5uL Sodium acetate

560uL cold ethanol

50 ml Conical Tubes

Methods

Chemical Extraction- Guanadiniumthiocyanate solution method

500uL of guanadinuimthiocyanate(GuSCN) solution was pipetted with a 100-1000uL pipette

into a sterile 1.5mL eppendorf tube. The following steps were used to prepare a group control.

The rest of the group also individually prepared 1 sample each, but only one control was needed

for the entire group.

A hair sample was pulled out of the head with a quick, smooth motion to ensure that the root was

intact. The hair was then cut with scissors to a length of approximately 3/8th of an inch being

sure to include the root tip. The hair/root sample was placed in the sample tube with the root

facing down. Both the sample and control were incubated at 56C on the heat block for one hour

under medium agitation.

The samples were then removed from the heat block. The hair sample was removed from the

sample tube and disposed of.


The tip of a 100-1000uL pipette tip was cut off and approximately 500uL of the

guanadiniumthiocyanate/ silica bead solution was pipetted into the control and sample tubes.

The tubes were then vortexed for 30 seconds and placed on ice for 15 minutes. The tubes were

removed from the ice and vortexed briefly at 5 minute intervals during the 15 minutes on ice.

After the 15 minutes on ice, the tubes were removed and quick-spinned on the mini centrifuge

for 15 seconds.

The supernatant was carefully removed by decanting in a trash container, and then the 20-200uL

pipette was used once there is little supernatant left. Care was taken not to dump or nick the

pellet.

The silica bead pellet was then re-suspended by adding 500uL of wash buffer and vortexing until

re-suspension. This was then quick-spinned on the mini-centrifuge for 15 seconds and the

supernatant was again removed by decanting and then pipetting.

200uL of ethanol were added to both tubes with a 100-1000uL pipette and vortexed for 15

seconds. These were then quick-spinned on the mini-centrifuge for 15 seconds. The supernatant

was again decanted and pipetted out of the tubes. The tubes were then air-dried by setting them

on the heat block until dried.

The pellets were then re-suspended by adding 250uL of sterile water and vortexing. Both the

sample and control were incubated on the heat block for one hour at 56C and stored for the

following lab session.

Physical Extraction- Chelex Method


Using a cotton-tip swab, the inside of the cheek was swabbed for 5 minutes. The cotton-tip swab

was then cut to a length of about 3/8th of an inch and placed inverted in a 1.5mL eppendorf tube.

500uL of Chelex solution was added to the sample tube and a control tube. A clean cotton-tip

swab was cut to a length of 3/8th on an inch and placed in the control tube. The rest of the group

also individually prepared 1 sample each, but only one control was needed for the entire group.

Both tubes were vortexed for 1 minute and incubated on the heat block for 1-2 hours at 56C

under medium agitation. These were then centrifuged on the bench-top centrifuge for 5 minutes

at 12 000 rpm. The cotton-swab tips were removed from the sample and control tubes. Both

tubes were centrifuged for 2 minutes on the bench-top centrifuge. The supernatant from each

tube was then transferred to sterile eppendorf tubes and stored for PCR amplification.

DNA Quantification and Purity

Both the chelex extraction method and guanadinium thiocyanate extraction method tubes were

taken out of storage. From each tube, 10uL of the solution was pipetted with a 2-20uL pipette

into 2 clean eppendorf tubes. These were labeled as un-pure sample and set aside.

The samples were then centrifuged for 1 minute and the guanadinium thiocyanate solution was

vortexed for 30 seconds. Half of each sample was pipetted into sterile eppendorf tubes. The

chelex tube contained 150uL and the guanadinium thiocyanate tube contained 75uL. To this,

10% v/v 3M sodium acetate was added which equals 7.5uL for the GuSCN tube and 15uL for the

chelex tube. These were then vortexed for 1 minute. 2.5X volume of cold ethanol was then

pipetted into each tube - 185uL in the GuSCN tube and 375uL in the chelex tube. These were

placed on ice for 30 minutes. Once this was done, the samples were centrifuged on the bench-top

centrifuge for 5 minutes at 13 000rpm and at 4C. The hinges of the tubes were placed on the


outside to help find the pellet. The supernatant was then decanted from each tube and a 2-20uL

pipette was used to remove the remaining supernatant. The pellets were air dried on the heat

block set at 37C to speed-up the drying process. Once dry, the pellets were re-suspended by

adding 150uL of sterile water to each and vortexing for 1 minute. Both tubes were then set in the

heat block at 37C for 15 minutes.

Once this was done, the un-pure sample tubes set aside earlier as well as the pure sample tubes

from the previous step were each diluted to 700uL. To the un-pure chelex and GuSCN tubes, a

1:70 dilution was carried out. This means that to 10uL of sample, 690uL of sterile water were

added with a 100-1000uL pipette. The purified samples contained approximately 150uL of

sample, so 100uL of each was pipetted into sterile eppendorf tubes. To each tube, 600uL of

sterile water was added with a 100-1000uL pipette. The remaining 50uL of sample in the chelex

and GuSCN tubes were stored for polymerase chain reaction amplification. Once the dilutions

were carried out, the four samples were pipetted into 1.5mL cuvettes and set in the

spectrophotometer at 260nm. The readings of each were recorded. The spectrophotometer was

then set to 280nm and the readings of the four samples were again recorded.

Results


Table 1 Dilution factor, 260nm and 280nm spectrophotometric readings, 260/280 ratio, and

DNA concentration of Chelex and Guanadinium thiocyanate(GuSCN) pre- and post-purification

Sample Dilution Factor 260nm Reading

280nm Reading

260/280 Ratio

[DNA]ug/mL

Pre-purification GuSCN

1:70(10uL sample:690uL H2O)

0.277 0.213 1.30 0.020

Pre-purification Chelex


0.003 0.004 0.75 0.002

Post-purification GuSCN


0.629 0.621 1.01 4.49

Post-purification Chelex


0.031 0.060 0.52 0.22

Note: See appendix 1.1 for sample calculations for the 260/280 ratio and for [DNA]

Table 2 Pellet and DNA observations for each step of experiment for Chelex, Guanadinium thiocyanate(GuSCN), and control tubes pre- and post-purification


Sample Step Pellet Supernatant Location of DNA Contents of Pellet

Contents of Supernatant or solution

Pre-purification GuSCN

After: incubation

Silica bead Pellet

clear In solution NA Crude extract of proteins and DNA

Quick spin 1 Silica bead Pellet

Clear Pellet DNA crude extract

Proteins and cellular debris







Air-dry + re-suspension

Silica bead Pellet

A bit cloudy In solution NA DNA crude extact

Chelex After: Incubation

No pellet A bit foamy In solution NA Crude extract of proteins and DNA

Centrifuge 5min

Pellet Clear Supernatant Proteins and other cellular debris

Crude DNA extract

Centriguge 2min

Pellet Clear Supernatant- observation of stringy DNA visible

Proteins and other cellular debris

Crude DNA extract

Chelex Control All Steps performed the same without addition of cheek cells

NONE CLEAR NONe NONE NONE

GuSCN Control All Steps performed the same without addition of hair sample

Pellet of Silica Beads

CLEAR NONE NONE NONE

Post-purification GuSCN

After: Centrifuge

White Pellet Clear Pellet DNA extract



NONE Clear In solution NA Purified DNA extract

Chelex After: Centrifuge

White Pellet Clear Pellet DNA Extract



NONE Clear In solution NA Purified DNA extract

Discussion


To extraction methods were studied in this lab. These include the guanidinium

thiocyanate(GuSCN)/silica bead method and the chelex method.

Guanidinium thiocyanate solution(GuSCN) is composed of guanidinium thiocyanate, tris-

hydrogen chloride, ethylenediaminetetraacetic acid, and triton X-100. Ethylenediaminetetraacetic

is used in the sample because of its property of binding metal ions(chelating agent).[10] Triton X-

100 is used to help lyse the cells in a sample and is also useful in protein purification. [9] Tris-HCl

is used as a buffer so that the pH of the solution is within range to prevent DNA degradation. For

the GuSCN/silica bead method as hair sample was used. This contained both the hair shaft and

the root tip. Two types of DNA are present in hair which includes chromosomal DNA and

mitochondrial DNA. Once the hair sample was added to the tube, GuSCN was added to lyse the

cells and dehydrate the DNA since it is a chaotropic agent. GuSCN is also used to denature

protein in the sample. This was also helped by the addition of heat at 56C for 1 hour. After

removal from heat, a solution of GuSCN/ silica beads was added to bind the DNA. Since DNA is

negatively charged, it binds to the positively charged silica beads. This is then placed on ice to

further help the DNA bind and centrifuged to precipitate DNA. A wash buffer composed of tris-

HCl, ethanol, NaCl, and EDTA was then added to remove solubilized proteins, RNA and metal

ions in the sample.[8] The supernatant containing the protein, metal ions, and RNA was then

removed. Ethanol was then added to further purify the sample since DNA is not soluble in

ethanol and therefore precipitates.

The chelex extraction method is a physical extraction and is a simple and cost effective way or

extracting DNA. It is composed of styrene divinlybenzene copolymers with paired

iminodiacetate ions. Because of the harsh environment of chelex, it is not suitable for highly

degraded or low concentrations of DNA. This harsh environment is cause by a pH between 10-


11 and high temperatures.[7] For this experiment, the inside of the cheek was swabbed to collect

cells. The DNA included in these cells was chromosomal and mitochondrial DNA. Since the

inside of the cheek is filled with bacteria, there might have been a small quantity of pathogenic

and bacterial DNA that would contaminate our sample. Once the tip was added to the Chelex

solution, it was heated for 2 hours at 56C. The heat and chelex caused the cells to lyse in the

sample thereby releasing the DNA. The negatively charged iminodiacetate ions in Chelex

chelate(bind) metal ions out of solution. Mg ions in the sample activate nuclease so removing the

ions prevents this activity.[7] The DNA is located in the supernatant and the resulting precipitate

is composed of metal ions.

An ethanol purification was also performed to further purify the GuSCN sample and to purify the

Chelex sample. Sodium acetate was added to both samples as a source of Na+ for the negatively

charged DNA to bind too. Cold ethanol is then added to precipitate the DNA out of solution.

DNA is insoluble in water because of ethanol’s low dielectric constant. Placing on ice further

improves DNA precipitation because compounds are more soluble at higher temperatures.

Centrifugation helps the suspended precipitate to settle at the bottom.

The main purpose of the extractions is to compare DNA yield. For pre-purification samples, the

GuSCN extraction method yielded a much higher concentration of DNA at 0.020ug/mL than the

Chelex extraction at 0.002ug/mL. This can be seen by the [DNA]ug/mL column readings in

Table 1. This is probably due to the fact that more sample was collected from the hair sample

because it was directly put in the tube compared to the cheek swab which most likely had fewer

cells available. Also, since Chelex is a harsh extraction due to the pH being between 10-11, alot

of the DNA could have been destroyed.[7] Post-purification, the GuSCN still had a higher

concentration of DNA than chelex.


Purification of the samples had a positive effect on improving DNA concentration. For GuSCN

the concentration rose from 0.020ug/mL to 4.49ug/mL. That is 223.5X increase which is

substantial considering the GuSCN extraction already has a purification step involved. The

chelex sample also increased, but only from 0.002ug/mL to 0.22ug/mL. That accounts for a

110.0X increase. It was expected that the Chelex sample would have a greater increase in yield

compared to the GuSCN sample since it had already been somewhat purified. An explanation for

this might be the fact that there was little DNA to begin with and that a lot of it was accidently

removed during the purification process. The ratios of DNA/protein or 260nm/280nm should

have increased after purification but they have decrease. This would indicate that more DNA

than protein has been lost in the purification process. Common errors could have been dislodging

the pellet and heating to long on the heat block and thereby damaging the DNA. It is expected

that some DNA would be lost in the extraction process, but one would hope that more protein

would be taken out of solution than DNA. The sodium acetate in the purification process not

only causes DNA to precipitate but it also cause proteins to salt-out.[12] Therefore the pellet could

contain a high concentration of protein. Therefore this would explain the lower ratio of 260/280

for the purified samples.

Conclusion

The purpose of the experiment was to compare and understand the two different extraction

methods which were guanidinium thiocyanate/ silica bead and chelex. Much has been learned

about the different methods, but the ratios of DNA post-purification could have been higher.

Purity of the DNA samples is something that should be looked into more to hopefully find better

ways to improve the ratio, however, user technique could also have been a factor that affected

yield.


References

[1] “Determination of DNA concentration by Spectrophotometric Estimation.” Accessed Nov.

13, 2010 http://homepages.bw.edu/~mbumbuli/molbio/labs/dna/index.html

[2]Rice, G. DNA Extraction. Montana State University. Accessed Nov.15, 2010

http://serc.carleton.edu/microbelife/research_methods/genomics/dnaext.html

[3] Capricci, J. “Comparison of two methods of DNA extraction from archaeological bone.”

Lakehead University 1996. pp15-17, 22-23

[4] Matheson, C. Lecture 4.2: Detection of DNA. Lakehead University, Presented Fall 2010

[5]Matheson, C. Lecture 3: Extraction Chemistry. Lakehead University. Presented Fall 2010.

[6] Student Guide to DNA Fingerprinting by PCR. Accessed Nov. 14, 2010

http://www.angelfire.com/mo3/disease/

[7] Chelex Extraction Process. Accessed Nov. 14, 2010

http://www.nfstc.org/pdi/Subject03/pdi_s03_m03_01.htm

[8] Chowdhury, E.D. et al. Rapid isolation of high quality, multimeric plasmid DNA using zwitterionic

detergent. Journal of Biotechnology. Volume 119, Issue 4, 10 October 2005, Pages 343-347

[9] TRITON X-100. Sigma Co.

http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/1/t8532pis.Pa

r.0001.File.tmp/t8532pis.pdf

[10] Ethylenediaminetetraacetic acid. University of Maryland Medical Center

http://www.umm.edu/altmed/articles/ethylenediaminetetraacetic-acid-000302.htm

[11] Mason, P.E. et al. “The hydration structure of guanidinium and thiocyanate ions: Implications for

protein stability in aqueous solution” Stanford University Medical Center. Stanford: October

2001.

[12] Promega. DNA Purification and Quantification. Accessed October 10, 2010

www.promega.com/education/unit004/DNA_Purification_Final.

http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/1/t8532pis.Pa

http://www.nfstc.org/pdi/Subject03/pdi_s03_m03_01.htm

http://www.angelfire.com/mo3/disease/

http://serc.carleton.edu/microbelife/research_methods/genomics/dnaext.html

http://homepages.bw.edu/~mbumbuli/molbio/labs/dna/index.html

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Extraction Methodologies