PCR PCR

Mr. S.GhoshMITS

Division of Biotechnology

The methods used by molecular biologists to study DNA have been developed through adaptation of the chemical reactions and biological processes that occur naturally in cells

Many of the enzymes that copy DNA, make RNA from DNA, and synthesize proteins from an RNA template were first characterized in bacteria. This basic research has become fundamental to our understanding of the function of cells and have led to immense practical applications for studying a gene and its corresponding protein.

As science advances, so do the number of tools available that are applicable to the study of molecular genetics.

Laboratory Tools and Techniques

PCR

The Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying relatively large quantities of DNA First described in 1985, Nobel Prize for Kary Mullis in 1993

The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the bacterium Thermus aquaticus that was discovered in hot springs

The primary materials, or reagents, used in PCR are:- DNA nucleotides, the building blocks for the new DNA - Template DNA, the DNA sequence that you want to amplify - Primers, single-stranded DNAs between 20 and 50 nucleotides long (oligonucleotides) that are complementary to a short region on either side of the template DNA - DNA polymerase, a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA

PCR

The cycling reactions : There are three major steps in a PCR, which are repeated for 20 to 40 cycles. This is done on an automated Thermo Cycler, which can heat and cool the reaction tubes in a very short time. Denaturation at around 94°C : During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example the extension from a previous cycle). Annealing at around 54°C :Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. If the primers exactly fit the template, the hydrogen bonds are so strong that the primer stays attached Extension at around 72°C :The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)

PCR

The different steps of PCR

PCR

Exponential increase of the number of copies during PCR

PCR

Every cycle results in a doubling of the number of strands DNA present

After the first few cycles, most of the product DNA strands made are the same length as the distance between the primers

The result is a dramatic amplification of a the DNA that exists between the primers. The amount of amplification is 2 raised to the n power; n represents the number of cycles that are performed. After 20 cycles, this would give approximately 1 million fold amplification. After 40 cycles the amplification would be 1 x 1012

PCR and Contamination

The most important consideration in PCR is contamination

Even the smallest contamination with DNA could affect amplification

For example, if a technician in a crime lab set up a test reaction (with blood from the crime scene) after setting up a positive control reaction (with blood from the suspect) cross contamination between the samples could result in an erroneous incrimination, even if the technician changed pipette tips between samples. A few blood cells could volitilize in the pipette, stick to the plastic of the pipette, and then get ejected into the test sample

Modern labs take account of this fact and devote tremendous effort to avoiding cross-contamination

Optimizing PCR protocols

While PCR is a very powerful technique, often enough it is not possible to achieve optimum results without optimizing the protocol

Critical PCR parameters:

- Concentration of DNA template, nucleotides, divalent cations (especially Mg2+) and polymerase

- Error rate of the polymerase (Taq, Vent exo, Pfu)

- Primer design

PCR can be very tricky

Primer design

General notes on primer design in PCR

Perhaps the most critical parameter for successful PCR is the design of primers

Primer selection Critical variables are: - primer length - melting temperature (Tm) - specificity - complementary primer sequences - G/C content- 3’-end sequence

Primer length

- specificity and the temperature of annealing are at least partly dependent on primer length- oligonucleotides between 20 and 30 (50) bases are highly sequence specific

- primer length is proportional to annealing efficiency: in general, the longer the primer, the more inefficient the annealing- the primers should not be too short as specificity decreases

Primer design

Specificity

Primer specificity is at least partly dependent on primer length: there are many more unique 24 base oligos than there are 15 base pair oligos

Probability that a sequence of length n will occur randomly in a sequence of length m is:

Example: the mtDNA genome has about 20,000 bases, the probability of randomly finding sequences of length n is:

n Pn

5 19.5210 1.91 x 10-2

15 1.86 x 10-5

P = (m – n +1) x (¼)n

Primer design

Complementary primer sequences- primers need to be designed with absolutely no intra-primer homology beyond 3 base pairs. If a primer has such a region of self-homology, “snap back” can occur- another related danger is inter-primer homology: partial homology in the middle regions of two primers can interfere with hybridization. If the homology should occur at the 3' end of either primer, primer dimer formation will occur

G/C content- ideally a primer should have a near random mix of nucleotides, a 50% GC content- there should be no PolyG or PolyC stretches that can promote non-specific annealing

3’-end sequence - the 3' terminal position in PCR primers is essential for the control of mis-priming- inclusion of a G or C residue at the 3' end of primers helps to ensure correct binding (stronger hydrogen bonding of G/C residues)

Primer design

Melting temperature (Tm)

- the relationship between annealing temperature and melting temperature is one of the “Black Boxes” of PCR

- a general rule-of-thumb is to use an annealing temperature that is 5°C lower than the melting temperature

- the goal should be to design a primer with an annealing temperature of at least 50°C

- the melting temperatures of oligos are most accurately calculated using nearest neighbor thermodynamic calculations with the formula: Tm = H [S+ R ln (c/4)] –273.15 °C + 16.6 log 10 [K+] (H is the enthalpy, S is the entropy for helix formation, R is the molar gas constant and c is the concentration of primer)

- a good working approximation of this value can be calculated using the Wallace formula: Tm = 4x (#C+#G) + 2x (#A+#T) °C

- both of the primers should be designed such that they have similar melting temperatures. If primers are mismatched in terms of Tm, amplification will be less efficient or may not work: the primer with the higher Tm will mis-prime at lower temperatures; the primer with the lower Tm may not work at higher temperatures.

The PCR Process—Reaction The PCR Process—Reaction ComponentsComponents

Typical components of a PCR include:◦DNA: the template used to synthesize new DNA

strands.◦DNA polymerase: an enzyme that synthesizes new

DNA strands.◦Two PCR primers: short DNA molecules

(oligonucleotides) that define the DNA sequence to be amplified.

◦Deoxynucleotide triphosphates (dNTPs): the building blocks for the newly synthesized DNA strands.

◦Reaction buffer: a chemical solution that provides the optimal environmental conditions.

◦Magnesium: a necessary cofactor for DNA polymerase activity.

HPCR Amplify DNA?HPCR Amplify DNA? One PCR cycle consists of a DNA denaturation

step, a primer annealing step and a primer extension step.

DNA Denaturation: Expose the DNA template to high temperatures to separate the two DNA strands and allow access by DNA polymerase and PCR primers.Primer Annealing: Lower the temperature to allow primers to anneal to their complementary sequence.Primer E xtension: Adjust the temperature for optimal thermostable DNA polymerase activity to extend primers.

PCR uses a thermostable DNA polymerase so that the DNA polymerase is not heat-inactivated during the DNA denaturation step. Taq DNA polymerase is the most commonly used DNA polymerase for PCR.

Mechanism of DNA SynthesisMechanism of DNA Synthesis

◦DNA polymerase extends the primer by sequentially adding a single dNTP (dATP, dGTP, dCTP or dTTP) that is complementary to the existing DNA strand

◦The sequence of the newly synthesized strand is complementary to that of the template strand.

◦The dNTP is added to the 3´ end of the growing DNA strand, so DNA synthesis occurs in the 5´ to 3´ direction.

InstrumentationInstrumentation

Thermal cyclers have a heat-conducting block to modulate reaction temperature. ◦Thermal cyclers are programmed to maintain

the appropriate temperature for the required length of time for each step of the PCR cycle.

◦Reaction tubes are placed inside the thermal cycler, which heats and cools the heat block to achieve the necessary temperature.

Thermal Cycling ProgramsThermal Cycling Programs

A typical thermal cycler program is:Initial DNA denaturation at 95°C for 2 minutes 20–35 PCR cycles: Denaturation at 95°C for 30 seconds to 1 minuteAnnealing at 42–65°C for 1 minuteExtension at 68–74°C for 1–2 minutesFinal extension at 68–74°C for 5–10 minutesSoak at 4°C

PCR OptimizationPCR Optimization

Many PCR parameters might need to be optimized to increase yield, sensitivity of detection or amplification specificity. These parameters include:

Magnesium concentrationPrimer annealing temperaturePCR primer designDNA qualityDNA quantity

Magnesium ConcentrationMagnesium Concentration

Magnesium concentration is often one of the most important factors to optimize when performing PCR.

The optimal Mg2+ concentration will depend upon the primers, template, DNA polymerase, dNTP concentration and other factors.◦ Some reactions amplify equally well at a number of Mg2+

concentrations, but some reactions only amplify well at a very specific Mg2+ concentration. 

When using a set of PCR primers for the first time, titrate magnesium in 0.5 or 1.0mM increments to empirically determine the optimal Mg2+ concentration.

Primer Annealing TemperaturePrimer Annealing Temperature

PCR primers must anneal to the DNA template at the chosen annealing temperature.

The optimal annealing temperature depends on the length and nucleotide composition of the PCR primers

The optimal annealing temperature is often within 5°C of the melting temperature (Tm) of the PCR primerThe melting temperature is defined as the temperature at

which 50% of complementary DNA molecules will be annealed (i.e., double-stranded).

When performing multiplex PCR, where multiple DNA targets are amplified in a single PCR, all sets of PCR primers must have similar annealing temperatures.

DNA QualityDNA QualityDNA should be intact and free of

contaminants that inhibit amplification. ◦Contaminants can be purified from the original

DNA source. Heme from blood, humic acid from soil and

melanin from hair◦Contaminants can be introduced during the

purification process. Phenol, ethanol, sodium dodecyl sulfate (SDS) and

other detergents, and salts.

DNA QuantityDNA Quantity

DNA quantityMore template is not necessarily better.

◦Too much template can cause nonspecific amplification.

◦Too little template will result in little or no PCR product.

The optimal amount of template will depend on the size of the DNA molecule.

Applications of PCRApplications of PCR

PCR and RT-PCR have hundreds of applications. In addition to targeting and amplifying a specific DNA or RNA sequence, some common uses include:

Labeling DNA or RNA molecules with tags, such as fluorophores or radioactive labels, for use as tools in other experiments.

Cloning a DNA or RNA sequenceDetecting DNA and RNAQuantifying DNA and RNAGenotyping and DNA-based identification

Labeling DNALabeling DNA

Labeling DNA with tags for use as tools (probes) to visualize complementary DNA or RNA molecules.◦Radioactive labels.

Radioactively labeled probes will darken an X-ray film.◦Fluorescent labels (nonradioactive)

Fluors will absorb light energy of a specific wavelength (the excitation wavelength) and emit light at a different wavelength (emission wavelength).

The emitted light is detected by specialized instruments such as fluorometers.

Quantitative PCRQuantitative PCRAvoids problems associated with the plateau

effect, which reduces amplification efficiency and limits the amount of PCR product generated due to depletion of reactants, inactivation of DNA polymerase and accumulation of reaction products.◦The result of the plateau effect is that the amount of

PCR product generated is no longer proportional to the amount of DNA starting material.

◦The plateau effect becomes more pronounced at higher cycle numbers.

Often performed in real time to monitor the accumulation of PCR product at each cycle.◦Real-time PCR allows scientists to quantify DNA

before the plateau effect begins to limit PCR product synthesis.

RT PCRRT PCR

28

THE PROBLEM

• NEED TO QUANTITATE DIFFERENCES IN mRNA EXPRESSION

• SMALL AMOUNTS OF mRNA– LASER CAPTURE– SMALL AMOUNTS OF TISSUE– PRIMARY CELLS– PRECIOUS REAGENTS

29

THE PROBLEM

• QUANTITATION OF mRNA– northern blotting

– ribonuclease protection assay

– in situ hybridization

– PCR

• most sensitive• can discriminate closely related mRNAs• technically simple• but difficult to get truly quantitative results using

conventional PCR

30

NORTHERN

target gene

internal control geneactin, GAPDH, RPLP0 etc

Ratio target gene in experimental/control = fold change in target gene fold change in reference gene

control expt

Corrected fold increase = 10/2 = 5

Standards Standards

same copy number in all cellsexpressed in all cellsmedium copy number advantageous

◦correction more accurate

31

32

0

200000000

400000000

600000000

800000000

1000000000

1200000000

1400000000

1600000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBERA

MO

UN

T O

F D

NA

110

1001000

10000100000

100000010000000

1000000001000000000

10000000000

0 5 10 15 20 25 30 35

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

CYCLE NUMBER AMOUNT OF DNA0 11 22 43 84 165 326 647 1288 2569 512

10 1,02411 2,04812 4,09613 8,19214 16,38415 32,76816 65,53617 131,07218 262,14419 524,28820 1,048,57621 2,097,15222 4,194,30423 8,388,60824 16,777,21625 33,554,43226 67,108,86427 134,217,72828 268,435,45629 536,870,91230 1,073,741,82431 1,400,000,00032 1,500,000,00033 1,550,000,00034 1,580,000,000

33

34

35

Linear ~20 to ~1500

36

Linear ~20 to ~1500

37

REAL TIME PCR

• kinetic approach

• early stages

• while still linear

www.biorad.com

38

2a. excitation filters

2b. emission filters

1. halogen tungsten lamp

4. sample plate

3. intensifier

5. ccd detector 350,000 pixels

39

SERIES OF 10-FOLD DILUTIONS

40

41

42

SERIES OF 10-FOLD DILUTIONS

43

SERIES OF 10-FOLD DILUTIONS

threshold

Ct

44

threshold = 300

45

46

STANDARD CURVE METHOD

47

PFAFFL METHOD

– M.W. Pfaffl, Nucleic Acids Research 2001 29:2002-2007

48

AFTER 1 CYCLE 100% = 2.00x 90% = 1.90x 80% = 1.80x 70% = 1.70x

CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY

0 1 1 1 11 2 2 2 22 4 4 3 33 8 7 6 54 16 13 10 85 32 25 19 146 64 47 34 247 128 89 61 418 256 170 110 709 512 323 198 11910 1,024 613 357 20211 2,048 1,165 643 34312 4,096 2,213 1,157 58313 8,192 4,205 2,082 99014 16,384 7,990 3,748 1,68415 32,768 15,181 6,747 2,86216 65,536 28,844 12,144 4,86617 131,072 54,804 21,859 8,27218 262,144 104,127 39,346 14,06319 524,288 197,842 70,824 23,90720 1,048,576 375,900 127,482 40,64221 2,097,152 714,209 229,468 69,09222 4,194,304 1,356,998 413,043 117,45623 8,388,608 2,578,296 743,477 199,67624 16,777,216 4,898,763 1,338,259 339,44925 33,554,432 9,307,650 2,408,866 577,06326 67,108,864 17,684,534 4,335,959 981,00727 134,217,728 33,600,615 7,804,726 1,667,71128 268,435,456 63,841,168 14,048,506 2,835,10929 536,870,912 121,298,220 25,287,311 4,819,68630 1,073,741,824 230,466,618 45,517,160 8,193,466

0

200,000,000

400,000,000

600,000,000

800,000,000

1,000,000,000

1,200,000,000

0 10 20 30

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

100% EFF

90% EFF

80% EFF

70% EFF

49

AFTER 1 CYCLE 100% = 2.00x 90% = 1.90x 80% = 1.80x 70% = 1.70x

AFTER N CYCLES:fold increase = (efficiency)n

CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY

0 1 1 1 11 2 2 2 22 4 4 3 33 8 7 6 54 16 13 10 85 32 25 19 146 64 47 34 247 128 89 61 418 256 170 110 709 512 323 198 11910 1,024 613 357 20211 2,048 1,165 643 34312 4,096 2,213 1,157 58313 8,192 4,205 2,082 99014 16,384 7,990 3,748 1,68415 32,768 15,181 6,747 2,86216 65,536 28,844 12,144 4,86617 131,072 54,804 21,859 8,27218 262,144 104,127 39,346 14,06319 524,288 197,842 70,824 23,90720 1,048,576 375,900 127,482 40,64221 2,097,152 714,209 229,468 69,09222 4,194,304 1,356,998 413,043 117,45623 8,388,608 2,578,296 743,477 199,67624 16,777,216 4,898,763 1,338,259 339,44925 33,554,432 9,307,650 2,408,866 577,06326 67,108,864 17,684,534 4,335,959 981,00727 134,217,728 33,600,615 7,804,726 1,667,71128 268,435,456 63,841,168 14,048,506 2,835,10929 536,870,912 121,298,220 25,287,311 4,819,68630 1,073,741,824 230,466,618 45,517,160 8,193,466

0

200,000,000

400,000,000

600,000,000

800,000,000

1,000,000,000

1,200,000,000

0 10 20 30

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

100% EFF

90% EFF

80% EFF

70% EFF

50

CYCLE AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA AMOUNT OF DNA100% EFFICIENCY 90% EFFICIENCY 80% EFFICIENCY 70% EFFICIENCY

0 1 1 1 11 2 2 2 22 4 4 3 33 8 7 6 54 16 13 10 85 32 25 19 146 64 47 34 247 128 89 61 418 256 170 110 709 512 323 198 11910 1,024 613 357 20211 2,048 1,165 643 34312 4,096 2,213 1,157 58313 8,192 4,205 2,082 99014 16,384 7,990 3,748 1,68415 32,768 15,181 6,747 2,86216 65,536 28,844 12,144 4,86617 131,072 54,804 21,859 8,27218 262,144 104,127 39,346 14,06319 524,288 197,842 70,824 23,90720 1,048,576 375,900 127,482 40,64221 2,097,152 714,209 229,468 69,09222 4,194,304 1,356,998 413,043 117,45623 8,388,608 2,578,296 743,477 199,67624 16,777,216 4,898,763 1,338,259 339,44925 33,554,432 9,307,650 2,408,866 577,06326 67,108,864 17,684,534 4,335,959 981,00727 134,217,728 33,600,615 7,804,726 1,667,71128 268,435,456 63,841,168 14,048,506 2,835,10929 536,870,912 121,298,220 25,287,311 4,819,68630 1,073,741,824 230,466,618 45,517,160 8,193,466

0

200,000,000

400,000,000

600,000,000

800,000,000

1,000,000,000

1,200,000,000

0 10 20 30

PCR CYCLE NUMBER

AM

OU

NT

OF

DN

A

100% EFF

90% EFF

80% EFF

70% EFF

51

52

SERIES OF 10-FOLD DILUTIONS

53

QUALITY CONTROL -QUALITY CONTROL -EFFICIENCY CURVES EFFICIENCY CURVES use pcr baseline subtraction (not curve fitting

default option) - see next slideset the threshold manually to lab standardcheck all melting curves are OKcheck slopes are parallel in log viewdelete samples if multiple dilutions cross line

together (usually at dilute end of curve)delete samples if can detect amplification at

cycle 10 or earliermake sure there are 5 or more pointscheck correlation coefficient is more than

1.990

54

55

Quality ControlQuality Controluse pcr baseline subtraction (not curve fitting

default option)set the threshold manually to lab standardcheck all melting curves are OKcheck slopes are parallel in log viewdelete samples if multiple dilutions cross line

together (usually at dilute end of curve)delete samples if can detect amplification at

cycle 10 or earliermake sure there are 5 or more pointscheck correlation coefficient is more than

1.990

56

57

tissue

extract RNA

copy into cDNA(reverse transciptase)

do real-time PCR

analyze results

58

tissue

extract RNA

copy into cDNA(reverse transciptase)

do real-time PCR

analyze results

Should be free of protein (absorbance 260nm/280nm)

Should be undegraded (28S/18S ~2:1)Should be free of DNA (DNAse treat)Should be free of PCR inhibitors

◦Purification methods◦Clean-up methods

59

OVERVIEWOVERVIEW

60

tissue

extract RNA

copy into cDNA(reverse transciptase)

do real-time PCR

analyze results

Importance of reverse Importance of reverse transcriptase primerstranscriptase primers

Oligo (dt)

Random hexamer (NNNNNN)

Specific

61

Reverse TranscriptionReverse Transcription

adds a bias to the results

efficiency usually not known

62

63

tissue

extract RNA

copy into cDNA(reverse transciptase)

do real-time PCR

analyze results

Importance of primers in PCRImportance of primers in PCR

specifichigh efficiencyno primer-dimersIdeally should not give a DNA signal

◦cross exon/exon boundary

64

Number GamesNumber Games

Nested PCRNested PCR

Hot Start PCRHot Start PCR

Multiplex PCRMultiplex PCR

RT PCRRT PCR

THANK U