PCR techniques, DNA polymerase and RNA-polymerase used in PCR, Role of Mg++ and NTPs., PCR techniques

and its multiple uses. The use of PCR in gene assembly: multiplex PCR, Overlap extension PCR. Real time quantitative PCR.

PCR in molecular diagnostics, application of RT-PCR in gene expression

Mitesh Shrestha

Polymerase Chain Reaction

• PCR – first described in mid 1980’s, Mullis Nobel prize in 1993

• An in vitro method for the enzymatic synthesis of specific DNA sequences

• Selective amplification of target DNA from a heterogeneous, complex DNC/cDNA population

• Requires

• Two specific oligonucleotide primers

• Thermostable DNA polymerase

• dNTP’s

• Template DNA

• Sequential cycles of (generally) three steps (temperatures)

Directional Synthesis

Polymerase Chain Reaction

• The basic steps in conducting a conventional PCR involves;

• Denaturation achieved by heating the reaction mixture to a temperature between 90-98° C such that the dsDNA is denatured into single strands by disrupting the hydrogen bonds between complementary bases

• Annealing achieved by cooling the reaction mixture to a temperature of 45-60° C such that the primers base pair with the complementary sequence in the DNA and the hydrogen bonds reform.

• Elongation/ Extension achieved by adjusting the temperature to 72° C which is ideal for polymerase allowing primers extension by joining the bases complementary to DNA strands, the polymerase continually adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template.

• This completes a first cycle another cycle is continued. As PCR machine is automated thermocycler the same cycle is repeated upto 30-40 times

PCR - before the thermocycler

8 BORING hours per PCR!

95º C5 min

35 times

55º C3 min

72º C5 min

heated lids

adjustable ramping times

single/multiple blocks

gradient thermocycler blocks

Thermocyclers

standard tube, volume, costevaporation & heat transfer concerns

thin walled tube, volume, cost evaporation & heat transfer

concerns

Step 1:DenaturationdsDNA to ssDNA

Step 2:AnnealingPrimers onto template

Step 3:ExtensiondNTPs extend 2nd strand

extension products in one cycle serve as template in the next

A simple thermocycling protocol

ann

ealing

94ºC 94ºC

55ºC

72ºC

4ºC

3 min 1 min

45 sec

1 min

∞ hold

Initial denaturation of DNA

1X 35X 1X

extensio

n

de

natu

ration

Basic Components of PCR

• Template DNA (0.5 - 50 ng) < 0.1 ng plasmid DNA, 50 ng to 1 μg gDNA for single copy genes

• Oligonucleotide primers (0.1 – 2.0 μM)

• dNTP’s (20 –250 μM)

• Thermostable DNA pol (0.5 – 2.5 U/rxn)

• MgCl2 (1 – 5 mM) affects primer annealing and Taq activity

• Buffer (usually supplied as 10X)Working concentrations

KCL (10 – 50 mM)

Tris-HCl (10 mM, pH 8.3)

NaCl2 (sometimes)

dNTPsTaq polymerase

Primers

DNA template

Buffer

+ +

A C T G

MgCl2

Magnesium Chloride (MgCl2 - usually 0.5-5.0mM)

Magnesium ions have a variety of effectsMg2+ acts as cofactor for Taq polymerase

Required for Taq to function

Mg2+ binds DNA - affects primer/template interactions

Mg2+ influences the ability of Taq pol to interact with primer/template sequences

More magnesium leads to less stringency in binding

MgCl2 (mM) 1.5 2 3 4 5

PCR ProblemsTaq is active at low temperatures

At low temperatures mis-priming is likely

• “Cheap” fixes

– Physical separation –”DNA-in-the-cap”

– Set up reactions on ice

• Hot-start PCR –holding one or more of the PCR components until the first heat denaturation

– Manually - delay adding polymerase

– Wax beads

– Polymerase antibodies

• Touch-down PCR – set stringency of initial annealing temperature high, incrementally lower with continued cycling

• PCR additives

– 0.5% Tween 20

– 5% polyethylene glycol 400

– betaine

– DMSO

“Cures” for mis-priming

Primer Design

1. Typically 20 to 30 bases in length

2. Annealing temperature dependent upon primer sequence (~ 50% GC content)

3. Avoid secondary structure, particularly 3’

4. Avoid primer complementarity (primer dimer)

5. The last 3 nucleotides at the 3` end is the substrate for DNA polymerase - G or C

6. Many good freeware programs available

Primer Dimers

• Pair of Primers5’-ACGGATACGTTACGCTGAT-3’5’-TCCAGATGTACCTTATCAG-3’

• Complementarity of primer 3’ ends5’-ACGGATACGTTACGCTGAT-3’

3’-GACTATTCCATGTAGACCT-5’

• Results in PCR productPrimer 15’-ACGGATACGTTACGCTGATAAGGTACATCTGGA-3’3’-TGCCTATGCAATGCGACTATTCCATGTAGACCT-5’

Primer 2

Rules of thumb for PCR conditions

Add an extra 3-5 minute (longer for Hot-start Taq) to your cycle profile to ensure everything is denatured prior to starting the PCR reaction

Approximate melting temperature (Tm) = [(2 x (A+T)) +(4 x (G+C))]ºCIf GC content is < 50% start 5ºC beneath Tm for annealing temperatureIf GC content ≥ 50% start at Tm for annealing temperature

Extension @ 72ºC: rule of thumb is ~500 nucleotide per minute. Use 3 minutes as an upper limit without special enzymes

“Special” PCR cycling protocolsTouchdown PCRStep-up PCRGradient cycling

Common PCR additives

BSA (usually at 0.1 to 0.8 µg/µL final concentration)Stabilize Taq polymerase & overcome PCR inhibitors

DMSO (usually at 2-5% v/v, inhibitory at ≤ 10% v/v)Denaturant - good at keeping GC rich template/primer strands from forming secondary structures.

Glycerol (usually at 5-10% v/v)Increases apparent concentration of primer/template mix, and often increases PCR efficiency at high temperatures.

Stringency enhancers (Formamide, Betaine, TMAC)Concentrations used vary by typeEnhances yield and reduces non-specific priming

Non-ionic detergents (Triton X, Tween 20 or Nonidet P-40) (0.1–1%)NOT SDS (0.01% SDS cuts Taq activity to ~10% of normal)Stabilize Taq polymerase & suppress formation of 2º structure

Typical PCR Temps/Times

hold4o C or 10 mM EDTA

Stop reaction

5 – 10 min

70o – 75o CFinal extension

0.5 – 2 min

70o – 75o CPrimer extension

0.5 – 1 min

45o – 65o CPrimer annealing

0.5 – 1 min

90o – 95o CDenature

1 – 3 min

90o – 95o CInitial denaturation

25 –40

cycles

Troubleshooting PCRNon-specific bands on your gel

Reagents, set-up Run negative controlTemplate concentration inappropriate Review guidelinesAnnealing temp too low Optimize by gradient PCRExtension time too short time for longer productsCycle number too high Review guidelinesPrimer design not appropriate specificityPrimer concentration too high Optimize by titrationNon-specific priming specificity, Hot StartMgCl2 concentration too high Optimize by titrationGC-rich template, 2° structure PCR additivesContaminating DNA Decontaminate work area:

use ARTs, wear gloves,pipettor, reagents, UV treat plastics

Troubleshooting PCRDiffuse smearing on your gel

Template concentration inappropriate Review guidelinesTaq concentration too high Optimize by titrationExtension time inappropriate Review guidelinesCycle number too high Reduce, review guidelinesPrimer design not appropriate specificityPrimer concentration too high Optimize by titrationNon-specific priming Use Hot StartMgCl2 concentration too high Optimize by titrationGC-rich template, 2° structure PCR additivesContaminating DNA Decontaminate work area:

use ARTs, wear gloves,pipettor, reagents, UV treat plastics

Troubleshooting PCRPoor or no amplification of bands

Problem with thermocycler, set-up, Run positive controlreagents

Enzyme concentration low ConcentrationAnnealing temp too low Optimize by gradient PCRExtension time too short Time for longer productsCycle number too low Review guidelinesPrimer design not appropriate SpecificityPrimer concentration too high Optimize by titrationNon-specific priming Specificity, Hot StartMgCl2 concentration too low Optimize by titrationGC-rich template, 2° structure PCR additives

Troubleshooting PCR

Prioritizing Approaches

“Pilot” error ( set-up errors common in the interim betweentraining with someone and working independently)

Template dilution error (concentration matters!)

Thermocycling parameter errors (temps/times)

Bad reagents (1. dNTPs, 2. primers, 3. Taq)

Unique template or template structure issues

Don’t get discouraged…validating PCRs can be tricky

Advantages of PCR

Quick

Reliable

Sensitive

Relatively easy

Specific

Disadvantage of PCRNeed for equipment

Taq polymerase is expensive

Contamination

False reactions

Internal control

Cross-reaction

Enrichment steps in (contaminated) samples

Capacity building needed

Unspecific amplification

Types of PCR• Assembly PCR• Allele-specific PCR

• Colony PCR

• Helicase-dependent Amplification

• Hot-start PCR

• Inverse PCR• Methylation-specific PCR (MSP)

• Multiplex PCR

• Nested PCR

• Overlap extension PCR

• Quantitative PCR (Q-PCR)• Reverse transcription PCR (RT-PCR)

• Touch-down PCR

ALLELE-SPECIFIC PCR

• A diagnostic or cloning technique which is based on single-nucleotide polymorphisms (SNPs) (single-base differences in DNA).

• It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP.

• PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.

COLONY PCR

• The screening of bacterial (E. coli) or yeast clones for correct ligation or plasmid products.

• Selected colonies of bacteria or yeast are picked with a sterile toothpick or pipette tip from a growth (agarose) plate. This is then inserted into the PCR master mix or pre-inserted into autoclaved water. PCR is then conducted to determine if the colony contains the DNA fragment or plasmid of interest

HELICASE-DEPENDENT AMPLIFICATION

• Similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles.

• DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.

HOT-START PCR

• Modified form of Polymerase chain reaction (PCR) which avoids a non-specific amplification of DNA by inactivating the taq polymerase at lower temperatures.

• In Hot start PCR specific antibodies are used to block the Taq-polymerase at lower temperature.

• An initial step at 95℃ is required for denaturing the antibodies linked to the active center of the enzyme. The anti-Taq antibodies reduce the Taq polymerase activity below 72℃, the optimal temperature at which the enzyme extends the primers. When the specific antibodies detach from Taq-polymerase, the amplification proceeds with greater specificity.

INVERSE PCR• Target DNA is lightly cut into smaller fragments of several kilobases by

restriction endonuclease digestion.

• Self-ligation is induced under low concentrations causing the phosphate backbone to reform. This gives a circular DNA ligation product.

• Target DNA is then restriction digested with a known endonuclease. This generates a cut within the known internal sequence generating a linear product with known terminal sequences. This can now be used for PCR (polymerase chain reaction).

• Standard PCR is conducted with primers complementary to the now known internal sequences

METHYLATION-SPECIFIC PCR (MSP)

• Methylation-specific PCR (MSP) is used to identify patterns of DNA methylation at cytosine-guanine (CpG) islands in genomic DNA.

• Target DNA is first treated with sodium bisulphite, which converts unmethylated cytosine bases to uracil, which is complementary to adenosine in PCR primers.

• Two amplifications are then carried out on the bisulphite-treated DNA: One primer set anneals to DNA with cytosines (corresponding to methylated cytosine), and the other set anneals to DNA with uracil (corresponding to unmethylated cytosine). MSP used in Q-PCR provides quantitative information about the methylation state of a given CpG island

Multiplex PCR

• Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment.

• In a multiplexing assay, more than one target sequence can be amplified by using multiple primer pairs in a reaction mixture. As an extension to the practical use of PCR, this technique has the potential to produce considerable savings in time and effort within the laboratory without compromising on the utility of the experiment.

• Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.

NESTED PCR

• Increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA.

• Two sets (instead of one pair) of primers are used in two successive PCRs.

• In the first reaction, one pair of primers “outer pair” is used to generate DNAproducts, which besides the intended target, may still consist of non-specificallyamplified DNA fragments.

• The product(s) are then used in a second PCR after the reaction is diluted with a setof second set “nested or internal” primers whose binding sites are completely orpartially different from and located 3' of each of the primers used in the firstreaction.

• The specificity of PCR is determined by the specificity of the PCR primers.

• For example, if your primers bind to more than one locus (e.g. paralog or commondomain), then more than one segment of DNA will be amplified.To control for these possibilities, investigators often employ nested primers toensure specificity

QUANTITATIVE PCR (Q-PCR)

• Used to measure the quantity of a PCR product (commonly in real-time).

• It quantitatively measures starting amounts of DNA, cDNA or RNA.

• Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample.

• Quantitative real-time PCR has a very high degree of precision.

• QRT-PCR methods use fluorescent dyes, such as Sybr Green, EvaGreenor fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time.

REVERSE TRANSCRIPTION PCR (RT-PCR)

• A PCR designed for amplifying DNA from RNA. Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites.

• If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR ( Rapid Amplification of cDNA Ends).

TOUCHDOWN PCR (STEP-DOWN PCR)

• A variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses.

• The annealing temperature at the initial cycles is usually a few degrees (3-5°C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5°C) below the primer Tm.

• The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.

Uses of PCR: Forensics

PCR’s ability to amplify even the smallest amount of DNA from samples

collected at a crime scene gives the method great power when used in criminal

forensics.

The DNA from body fluid, hair, or other tissue samples is amplified to create a

nearly unique pattern for each individual. This pattern can then be compared to

suspects in the case.

The infamous OJ Simpson case was the first one in which the technique of

PCR became widely publicized.

Uses of PCR: GMO Food Detection

Genetically-modified foods (GMO foods) are widely grown in the USA and other

countries.

For various reasons, some countries require exporters to indicate the

percentage of GMO content in grain and food shipments.

PCR can be used to accurately measure the exact quantity of genetically-

modified food in a shipment, by “looking” at the DNA that makes up the food!

Uses of PCR: Paternity Testing

PCR’s power at identifying individual genetic makeup has made it invaluable for

use in paternity testing.

By amplifying specific DNA fragments from parents or close relatives, it is

possible to reconstruct relatedness between individuals.

PCR can not only identify relationships between people today, but can also be

used to identify historical family relationships!

Uses of PCR: Archaeology

PCR has been used for many scientific studes in the field of

archaeology:

Reconstructing the Dead Sea Scrolls.

Identification of paint pigments in cave paintings.

Determining relatedness between individuals in ancient ossuaries.

Constructing dinosaurs from blood preserved in amber specimens. (!)

Uses of PCR: Disease Diagnosis

PCR is now invaluable in modern disease diagnosis.

PCR can identify disease-causing organisms much earlier than other methods,

since it looks for the DNA of the organism itself, not its proteins or its effect on

our immune system.

PCR has even been used to diagnose diseases of the past, by amplifying

minute amounts of disease-related DNA in preserved specimens.

Uses of PCR: Disease Treatment

PCR can not only be used in disease diagnosis, but also as an aid in the

treatment of diseases.

For example, real-time PCR is used to directly monitor the amount of HIV virus

in patients suffering from infection. By monitoring the amount of virus present,

the drug therapy can be continually adjusted to maximize virus suppression.

Uses of PCR: Wildlife Conservation

Because PCR can be used to identify not only individuals, but also can

differentiate between species, it is often used in wildlife conservation research.

PCR can be used to monitor trade in products made from endangered species.

PCR can be used to monitor ecosystems for the presence of certain species.

PCR can be used even to monitor and identify indvidual animals!

The Human Genome Project has identified tens of thousands of genes in the

human genome. A key questions is: what do these genes do? Part of the

answer comes from determining when the genes are turned on and off, and

what affects the level of gene expression. Quantitative PCR is a key

component of determining the levels of gene expression, and is a critical tool

in cancer research, disease studies, and developmental biology.

RNA

DNA Enzymes

GENEX AnalysisBiology

Uses of PCR: Basic Research

Useful Links

• http://www.dnai.org

• https://www.dnalc.org/resources/animations/pcr.html

• https://bitesizebio.com/19922/the-a-z-of-pcr-variants/

• https://www.ncbi.nlm.nih.gov/probe/docs/techpcr/

Assignment• Write short notes on different types of PCR. [8]

• Differentiate between Reverse Transcriptase PCR and Real Time PCR. [3]

• Cellular replication uses RNA primer while PCR uses DNA primer. Why? [3]

• Can PCR be performed with single primer? [1]

• During second strand synthesis from product obtained after RT-PCR, which of the primer gets utilized first? [3]

• What is the role of MgCl2 in PCR? [1]

• Explain in brief about various problems that occurs during PCR and their corresponding solutions. [3]

• What are the various considerations to be made during primer design? [3]