Biol 3061 Pcr

Dr. Nigel Austin

http://www.ncbi.nlm.nih.

gov/books/NBK7571/

http://www.ncbi.nlm.nih.

gov/books/NBK21128/

http://www.ncbi.nlm.nih.gov/books/NBK7571/




To familiarize students with the PCR technique and how its performed.

To illustrate how PCR is performed and how its results are analyzed.

To illustrate the importance of the PCR technique via its many applications.

What is PCR?

Requirements

Steps in PCR

Visualization of Products

Applications

Limitations

Polymerase: DNA polymerase (enzyme) ◦ DNA polymerase duplicates DNA

◦ Before a cell divides, its DNA must be duplicated

◦ Chain Reaction: The product of a reaction is used to amplify the same reaction

◦ Results in rapid increase in the product

Polymerase Chain Reaction (PCR) uses principles of DNA replication to exponentially copy or amplify genes or DNA segments

Sequence to be copied called “target DNA/sequence”

Viral RNA can also be used

Amplified fragment called an amplicon

1. Template DNA ◦ The target DNA usually originates from the

cells of an organism ◦ Genomic DNA is extracted from the organism

which contains the target sequence ◦ whole cells can also be used ◦ Strands must be separated ie single stranded

for amplification to occur

…TCGGGAGGTCCTGTCCGACGTAGTCTTCTCCGGTAGTTCGTCCAGACAAGGTTCCCGGAA… 3’ 5’

…AGCCCTCCAGGACAGGCTGCATCAGAAGAGGCCATCAAGCAGGTCTGTTCCAAGGGCCTT… 3’ 5’

Target DNA

2. dNTPs ◦ Raw materials necessary

to synthesize DNA ◦ 4 dNTPs – for each base

found in DNA

T

C

A

A

C

T

A

T

G C

T C G

3. Primers ◦ Short sequences of chemically synthesized DNA (10-30

bases)

◦ Bind to the ss template DNA (or RNA) ◦ 2 primers used, usually called forward (F) and reverse (R)

◦ F & R primers flank and define the target DNA (gene) to be copied

◦ Serve as recognition sites for the polymerase

GGGAGGTCCT 3’ 5’ GTTCCAAGGG

5’ 3’

4. Polymerase Enzyme ◦ Thermostable enzyme which synthesizes new DNA

◦ Recognizes primers as start point for synthesis

◦ Synthesizes new strand in 5’ to 3’ direction

◦ Requires buffer and MgCl2 to function

5. PCR Reaction Buffer ◦ Generally contains:

0-50mM Tris-HCl pH 8.3

up to 50mM KCl

BSA

◦ Stabilize polymerase and the amplicons during PCR process

6. MgCl2 – polymerase functioning

Thermal cycler/PCR Machine ◦ uses heat to melt (separate)

template DNA and anneal primers to template DNA

◦ Electronically controlled, capable of changing temperatures quickly and in a cyclic manner

◦ Inventor: Kary Mullis in 1983

got $10,000 for his invention

Nobel prize in 1993

Different protocols for extracting genomic DNA

DNA should not be contaminated with spurious DNA, carbohydrates, phenols, or other compounds as these would inhibit DNA amplification.

PCR components Amount

Template DNA (5-200 ng)

1 mM dNTPs (200 uM final)

10 X PCR buffer

25 mM MgCl2 (1.5 mM final)

20 uM forward primer (20 pmoles final)

20 uM reverse primer (20 pmoles final)

5 units/uL Taq DNA polymerase (1.5 units)

Water

Final Volume

3 uL

10 uL

5 uL

3 uL

1 uL

1 uL

0.5 uL

26.6 uL

50 uL

In a thin wall Eppendorf tube assemble the following:

PCR occurs in a 3 step process called a cycle

1. Denaturation ◦ Occurs at approx. 90-95C, 1min

◦ The H-bonds in isolated DNA is denatured & made ss

◦ ss DNA serves as template for DNA cloning

Heating separates the double stranded DNA ◦ Denaturation

Slow cooling anneals the two strands ◦ Renaturation

Heat Cool

2. Annealing ◦ Temperature quickly reduced to about 40-65C

◦ Allows primers to bind to complimentary regions at the beginning and end of target sequence of target DNA in ssDNA, 45s

◦ 2 primers reqd, 1 for each strand

◦ Annealing temp. dependent on the length & base sequence of primer

◦ Allows primer to bind to template at high specificity



GGGAGGTCCT 5’ 3’

GTTCCAAGGG 5’ 3’ T C

A

A

C

T

A T G

C

T C

G

3. Elongation ◦ Taq recognises primer and synthesises

complimentary strand using dNTPs ◦ 70-75C optimum, temp for taq polymerase

activity, 2min

T

G



GGGAGGTCCT 5’

GTTCCAAGGG 5’

T C C G

A

A

C

T

A G T C

C

T

C G G

The above steps is referred to as a cycle.

At least 30 cycles are done

Each cycle increases the copy no. exponentially

Fragments are resolved on either agarose/ polyacrylamide gels

Cycles Copies

1 2

2 4

4 16

10 1,024

15 32,768

20 1,048,576

25 33,554,432

30 1,073,741,824

Calculates the predicted yield of DNA

PCR product yield = (input target amount) x (1 + % efficiency) cycle number

L 1 2 3 4 5 6 7 8 9 10 11

L 1 2 3 4 5 6 7 8 9 10 11

Molecular Identification Sequencing Genetic Engineering

• Molecular Archaeology • Molecular Epidemiology • Molecular Ecology • DNA fingerprinting • Classification of organisms • Genotyping • Pre-natal diagnosis • Mutation detection • Paternity testing • Sex determination • Detection of pathogens

• Bioinformatics • Genomic cloning • Genome Projects

• Site-directed mutagenesis • Gene expression studies

Primers are necessary ◦ To synthesize primers, knowledge of the

DNA sequence of interest must be known ◦ PCR cannot be used to study/amplify

fragments of genes never studied before or be used in areas of the genome where no sequence info is available

◦ **Exception- Arbitrarily Primed PCR (AP-

PCR), RAPDs or PCR where small known sequences are added to fragments to which primers are then made – AFLP

Size of amplified Fragment (amplificon) ◦ PCR can clone fragments up to 5 kb without much

difficulty, 40kb with some modification

◦ Some applications such as genome sequencing requires fragments>100kb to be amplified, therefore PCR cannot be used

Identify the requirements for PCR

Identify the steps in PCR?

Calculate the predicted yield of PCR

Interpret a PCR temperature profile

Interpret a PCR efficiency profile

Identify limitations of this technology

Identify applications and uses of this technology?

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Biol 3061 Pcr