Basics of Polymerase Chain Reaction Technology

A. Fessehaie, PhDFessehai@iastate.eduSeed Science Center, Ames, IA, 50011Iowa State University

GT - WORKSHOP Feb. 5, 2010

Overview■ Polymerase Chain Reaction - What is it?

■ Method Development

◘ Sample collection and processing◘ Nucleic acid isolation◘ PCR amplification ● PCR types ● Enzyme type● Primer Design and specificity● Selection of procedure parameters

◘Amplification detection and confirmation● Gel electrophoresis

Overview Cont.◘ Optimization/Troubleshooting

◘ Method sensitivity, precision and recovery

◘ Limitation of conventional PCR

◘ General Considerations

Polymerase Chain Reaction (PCR)

What is a polymerase?A polymerase is a naturally occurring enzyme, a biological macromolecule that catalyzes the formation and repair of DNA (and RNA)

What is PCR?

◘Though the simplest and most convenient way to define PCR is as a technique, such compartmentalizingeliminates the history of PCR’s invention, therebycovering over the contingent manner of its emergenceand the practices and subjects required to make it work.

◘The next simplest answer is to name an individualas the inventor of the concept. The obviouscandidate is Kary B. Mullis, who was awarded the1993 Nobel Prize for chemistry for PCR.

“The Molecule of the Year” or “a Technique”

◘ However, this terrain is contested. Other scientists, including, Henry Erlich, Norman Arnheim, Randall Saiki, Glen Horn, Corey Levenson, Steven Scharf, Fred Faloona and Tom White, were instrumental in making PCR work.

◘A third argument holds that PCR did not exist until it was made to work in an experimental system. In this view, merely thinking of a concept is not sufficient; a scientific advance must include creating a way to show that the concept has successfully been put into practice.

What is PCR? Cont.

Paul Rabinow -- Berkeley, Calif., 1998

1. Paul Rabinow -- Berkeley, Calif., 1998 http://sunsite.berkeley.edu/PCR/whatisPCR.html

2. Foundations of PCR - A. Basic Research (Seminal reports of PCR Research/Discovery)

http://sunsite.berkeley.edu/PCR/foundationalPCR.html#anchor1239949

3. Guyer R. L; Koshland D. E ,Jr. The Molecule of the Year.Science, 1989 Dec 22, 246(4937):1543-6.

4. EPA- Quality Assurance/Quality Control Guidance for Lab performing PCR analysis on Environmental Samples, Oct 2004

References

Method Development◘ Equipments- Laminar-Flow Hoods; Thermocycler; Centrifuges,

- Gel electrophoresis chambers; Power supply

- Refrigerators, Ultra-low freezers; Incubators

- Spectrophotometer etc…

◘ Disposables - Pipette tips; Gloves; PCR tubes

Sample Collection and Processing

◘ Sample collection and transport conditions impact the results data of PCR analysis.

◘ Labs should develop a detailed sampling and processing SOP for each new method.

Goal is to isolate the organism or DNA of interest from environ. matrix, while minimizing co-purification of potential contaminants and components of the matrix that may inhibit PCR.

◘ Reagents□ Prepared In-house

● CTAB; SDS-based buffers

□ Commercially prepared

● Commercially available Kits

Nucleic acid isolation Methods

Nucleic acid quantity and quality

● Ethidium bromide stained gel image

● Spectrophotometric data - UV absorbance (λ260 /λ280) ratios

DNA ~ 1.8-2.0RNA ~ 1.9-2.1

Gel image showing total RNA extract from spinach (S) and Arabidopsis (A) leaf tissues fractionate by electrophoresis in 3% agarose gel stained with 0.5 µg/ul ethidium bromide (Keting CHEN, Horticulture Dept. ; Feb 21, 2008

A S A S

28S

18S

Quantity and quality of total genomic DNA determined with an NanoDrop-1000

Spectrophotometric estimation of DNA

● UV Spectrophotometric data - UV absorbance (λ260 /λ280) ratiosDNA ~ 1.8-2.0RNA ~ 1.9-2.1

PCR Amplification Developing methods to analyze environ. samples by PCR,

◘ Select the type of PCR that is most appropriate for analysis

◘Assess the performance of PCR amplification step separately

◘ then as part of the entire analytical method.

PCR Type - Five common designs or types□ Conventional PCR □ Real-time PCR □ Multiplex PCR □ Reverse transcription (RT)-PCR□ Nested PCR

Conventional PCR

Conv. PCR uses thermostable DNA polymerase to amplify DNA sequence of interest

First step - denaturation, the sample is heated to separate the two strand (15-60s)

Followed by annealing step, in which the reaction temp. lowered allowing the primers to bind to the separated single strands of the template DNA (30-60s)

The last step is elongation, during which the temperature is raised, allowing specific enzymes to make the complementary copy of the template nucleic acid

◘ Real-time PCR detects and measures the amplification target DNA as they are produced

◘ Unlike, conv. PCR, real-time PCR uses an oligonucleotide probe labeled with fluorescent dyes or an alternative chemistry, and a thermocycler equipped with the ability to measure fluorescence.

Real-time PCR

◘ Modification of conventional PCR in which two or more different PCR products are amplified

◘ While multiplex PCR provides a potential time saving by allowing simultaneous detection of multiple targets, significant optimization is required.

Multiplex PCR

◘ RT-PCR is used to amplify RNA target sequences, mRNA, viral RNA genome.

◘ This type of PCR involves an initial incubation of RNA sample with a reverse transcriptase enzyme and a DNA primer.

◘ DNA primers commonly used include oligos dT (an oligos consisting of only thymidine residues), random hexamers (primer made of six random nucleotides), or a specific primer.

Reveres Transcription (RT)-PCR

◘ Oligo dT will hybridize to the poly A tail of messenger and - viral RNAs and prime DNA from the 3’ end of the RNA molecule.

◘ Random hexamers work with any RNA, but require an extra initial incubation at 25°C

◘ Specific primers can be either the PCR primers that hybridize to the RNA at the 3’ side of the amplification region or a primer that hybridize

◘ RNAs inhibitor is added to RT-PCR rxns to prevent degradation of RNA target

Reveres Transcription (RT)-PCR

Nested PCR□ Conventional PCR with a second

round of amplification using a different set of primers

□ The second set of primers is specific to a sequence found within the initial conv. PCR amplicon.

□ Second amplifications step with nested primer set results in reduced background

□ Nested PCR increases sensitivity and specificity of PCR.

http://www.pcrstation.com

◘ Taq DNA polymeraseIsolated from thermophilic bacterium Thermus aquaticus

◘ HotStart DNA polymerasesEnzyme activity muted by antibody or chemical

◘ USB HotStart-ITUses single-stranded DNA binding protein to sequester primers at lower temp., making them unavailable for extension by polymerase

Enzyme type

◘ 18-24 bases long◘ No internal secondary structure◘ GC content should be between 40-70%◘ Not complementary to each other at the 3’ ends

(Primer-dimer formation)◘Avoid runs of identical nucleotides, spec. of 3 or

more Gs or Cs at the 3’ end◘ Melting Temperature (Tm) that allows annealing

temp. of 58-60°C◘ No hairpin loops with an energy of -0.5 kcal/mol or

less

Primer Design and Specificity

□ Thermal Cycling Conditions

□ Reaction Volumes

□ Primer and Template Concentration

□ PCR Reagents and Master Mix preparation

Selection of Procedure Parameters

PCR results must be confirmed.□ Confirmation that the specific target was

amplified can be accomplished during PCR using quantitative real-time probe-based hybridization or

□ following PCR using gel electrophoresis

Amplicon Detection and Confirmation

□ GE - the most common method used to detect products of conventional PCR.

□ Electrophoresis is the separation of charged molecules in an electrical field.

□ Movement of the nucleic acid through the gel is dependent on electric charge.

□ Separation is dependent on shape and size of amplicon.

Gel Electrophoresis

□ Nucleic acid molecules carry a uniform negative charge throughout their length, and the shape of all amplified DNA is linear

□ Hence, the major factor resulting in the separation of amplified DNA is size

□ The most common medium used for separation of DNA is agarose – a linear polymer that forms a solid gel of uniform density.

Gel Electrophoresis

□ To visualize DNA, gels are stained with fluorescent dyes (Ethidium bromide or SYBR Green I).

□ Various running dyes can be added along with the DNA in a loading buffer to monitor DNA migration through the gel.

□ Many different buffers and concentrations matrices are used to make and run gels.

□ Different combinations can provide more or less resistance to the DNA being separated to achieve the desired resolution and separation.

Gel Electrophoresis

PCR optimization needed?

Reaction Components Volume Final Conc.10X DNA polymerase buffer 5µl 1X

25 mM MgCl2 3µl 1.5mM10 µM dNTPs 2µl 0.2mM

25 µM Upstream primers 0.6µl 0.3µl 25 µM Downstream primer 0.6µl 0.3µlTaq DNA polymerase* (5U/µl) 0.25µl 0.025 U/µlTemplate DNA (1-5 ng/µl) 2µl -Nuclease-free water to a final vol. 50 µl -

* = DNA polymerase of your choice

Forward Primer (nM)Reverse Primer (nM) 25 . . 900

25 (25/25) x x x. x x x x. x x x x

x + + x

. x x x x900 x x x x

Primer optimization matrix

+ = Optimal PCR primer pair concentrations

I) DNA isolation(SDS buffer, Chow and Käfer (1993))

II) Conduct conventional PCR using DNA samples listed below:

i) DNA extracted from pure cultures of Sporisorium holic-sorghi ii) DNA extracted from maize leaf tissues spiked with Sporisorium

holic-sorghi sporesiii) DNA extracted from Sporisorium holic-sorghi-infected maize stalk

Sporisorium holic-sorghi-specific PCR assay (Xu et al. 1999)

Sporisorium holic-sorghi-infected maize stalk

DNA isolation:

~ 100 mg maize contaminated with S. holic-sorghi was scrubbed from stalk for DNA extraction

Quantity and quality of total genomic DNA was determined with an NanoDrop-1000

Spectrophotometric estimation of DNA

Quantification of DNA extractsusing a NanoDrop-1000

Module: Nucleic AcidPath: 10 mmSoftware: 3.5.2Sample ID ng/ul 260/280S. holic-sorghi DNA from pure teliospores 473.86 1.9S. holic-sorghi spores-spiked miaze leaf tissues 377.38 1.89S. holic-sorghi-infected miaze stalk 111.89 1.8

-- Normalize DNA to 5 ng/μl and use 2 μl in 25 μl PCR reactionmixture

PCR components ul/25 ul x rxns Vol (ul) Final Conc.

10X PCR Buffer 2.5 5 12.5 1X

25 mM MgCl2 1.25 5 6.25 1.25 mM

10 mM dNTP mix 2 5 10 0.2 mM of each dNTP

SR 1 F (25 uM) 0.3 5 1.5 0.3 uM

SR 1 R (25 uM) 0.3 5 1.5 0.3 uM

Taq polymerase HotStart (5U/ul) 0.125 5 0.625 0.025 U/ul

DNA (5 ng/ul) 2

water 16.525 5 82.625

25 115

Preparing the PCR reaction mix Thaw all required reagents completely and put them on ice; Mix all reagents well by inversion and spin them down prior to pipetting

EPA, United State Environ. Agency/ Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples (2004)

Thermal Cycling Conditions for PCR

■ PCR amplification of Sporisorium holic-sorghi DNA (Head smut of maize)

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SR1 SR3

◘ Touch-down PCRAnother modification of conv. PCR that can reduce non-specific amplification.

□ False positive can result from the non-specific amplification of cross-reacting DNA.

Detection of Non-specific PCR amplification

Cycle 94°C 54°C 72°C 4°C1 3 min - - -45 45s 45s 1 min 45s1 - - 9 min hold

PCR conditions:

beta-tubulin gene product

PCR amplification of ITS

● Non-specific amplification

Conventional PCR

■ Target 2/Anneal. 54°C

■ Target 1/Anneal. Temp. 58°C

● Specific PCR products

Number of cycles 96 94 58 57 56 54 72 4 1x 3' - - - - - - 4x 45' 45' - - - 1'30" 4x 45' 45' 1'30" 4x 45' 45' 1'30" 33x 45' 45' 1'30" 1x - - - - - - 9' Hold

Touchdown PCR

● PCR modification to reduce nonspecific amplification

PCR amplification of ITS

beta-tubulin gene product

Primer pair 2 (nM)Primer pair 1 (nM) 25 . . 900

25 (25/25) x x x. x x x x. x x x x

x + + x. x x x x

900 x x x x

Multiplex Primer optimization matrix

1 KB A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 1 KB

126 bp

70 bp

Figure 2. Data generated by PCR amplification with purified bacterial DNA. Products were separated on 3% agarose gel. The 126bp DNA fragment was amplified from bacterial genomic DNA. No PCR product was amplified from other non-target bacterial DNA. With template DNA from corn, a 70bp taxon-specific DNA fragment was amplified.

Upon optimization of individual components of the method, the entire method should be evaluated to determine the method’s limit of detection (sensitivity), precision, and recovery.

Method Sensitivity

□ Detection Limits of PCR procedure

● For +/- methods, the detection limit is the minimum conc. of analyte that produce a positive response with a given level of confidence.

Method SensitivityDetection Limits (DL) of PCR procedure and of the entire method

● DL of both methods are determined by analysis of triplicate, seeded samples containing 10-fold serially diluted target organism or target DNA.

Method Validation

● Specificity

● Detection limits

● Inter- and intra lab precision

● Recovery

● Simple and inexpensive● Require post-PCR analysis● Measurement of products by gel electrophoresis● Discrimination of different DNA fragments by

molecular weight ● Use mutagenic labeling reagents (Ethidium bromide,

SYBR Green) ● Do not provide sequence information● Could provide byproducts arising from the

amplification of non-specific sequences with thesame length of the desired fragment, leading to falsenegative results

Conventional PCR methods

● Post-PCR processing

● Size-based discrimination

● Non-automated

● Use carcinogenetic reagent (Ethidium bromide)

● PCR products do not necessarily reflectthe amount of DNA originally present in asample

Limitation of conventional PCR

PCR – General Considerations● Template

- Quality influences performance of PCR- Integrity of DNA (use ONLY DNA of high molecular weight)- Amount of template in a reaction strongly influences PCR

outcome

● Primers- 18-24 bases long- No internal secondary structure- GC content should be between 40-60%- Not complementary to each other at the 3’ ends (Primer-dimers)- Avoid runs of identical nucleotides, spec. of 3 or more Gs or Csat the 3’ end

- Melting Temperature (Tm) that allows annealing temp. of 58-60°C

● Optimal annealing temps. often higher than the Tm of the primers; have to be determined empirically.

● Mg2+ concentration - Wide range 1.5-3 mM- Forms soluble complex with dNTPs to produce the actual substrate that the polymerase recognizes

● dNTPs - Pre-mixed balanced PCR grade dATP, dGTP, dCTP, dTTP (1.5 mM with 200 dNTPs @ a conc. of 200 µM each)

● Choice of DNA polymerase - Taq DNA polymerase; (0.5 -2.5 U/50 ul rxn volume)

● pH - Buffer supplied with the Taq DNA polymerase is sufficient for optimal results

PCR – General Considerations

Thank You

DNA LabSeed Science Center

Iowa State University

DiagnosticsSeed Health Testing

GMO

R&DDNA Purification/Sequestration

Develop/establish PCR assays

CollaborationsSeed Pathol., PLP, Hort.,

Civil Eng., Agronomy

ACTIVITIES

PCR detection of Acidovorax avenaesubsp. citrulli in in cucurbit seeds

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PCR detection of Tobamovirus groups in solanaceous seeds

● Positive control was positive and easy to detect● Product size is 370 - 400bp

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400bp

100bp

50 300 … 90050 50/50 300/50 … 50/900

300 300/50 300/300 ... 300/900

450 450/50 450/300 ... 450/900

… ... ... ...

900 900/50 900/300 … 900/900

Table 1. Primer Titration Matrix

Primer pair 1 (nM)Pr

imer

pai

r 2 (

nM)