Davidson Day Ateh

Neuroscience Centre, Institute of Cell and Molecular Sciences Barts and The London School of Medicine and Dentistry

Queen Mary University of London

Experimental Neuropathology Module - November 2007

Intercalated Experimental Pathology BSc

PCR, Theory and Applications

DNA

DNA Replication In Vivo

DNA replication occurs during cell division

DNA Polymerase facilitates replication

• Different types of DNA polymerase (e.g. I, II & III and those involved in DNA repair)

• RNA polymerase for transcription

• High fidelity DNA synthesis is due to proof reading (only one error per 1 109 nucleotides)

Or in vitro DNA replication

Polymerase Chain Reaction

• A DNA polymerase (Taq) is used to make many copies of a short length of DNA defined by primers in a test tube

Thermus Aquaticus was discovered in the Yellowstone (USA) hotsprings in the 1960’s

and thrives at around 72°C

• Taq DNA polymerase works optimally at 72°C

• Critically it is not denatured at 94°C

(thermostable)

• Revolutionised by Kary Mullis in the 1980’s whilst he was working for a biotechnology company

• He received the 1993 Nobel Prize in Chemistry for his work

ONE OF THE MOST IMPORTANT MOLECULAR BIOLOGY TECHNIQUES

• PCR originally a slow, labour intensive process that required the addition of fresh DNA Polymerase every replication round

• Gene cloning (recombinant DNA techniques) developed in the 1970’s

PCR Mix

• Template DNA

• Buffer (with Mg2+)

• Nucleotides (dNTPs)

• Taq DNA Polymerase (or other thermostable DNA polymerase)

• Primers

Double Stranded DNA

Single Stranded DNA

Denaturing DNA template

Complimentary binding forward and a reverse

primers (20-30 oligonucleotides)

PCR Mechanisms

Melting

94 oC

Melting

94 oC

AnnealingPrimers

50 oC

Extension

72 oCT

empe

ratu

re

100

0

50

Time

30x

5’3’

3’5’

3’5’

5’

5’3’5’

3’5’

5’

5’

5’

5’3’

3’5’

3’5’

5’3’

5’3’

5’

PCR

Melting

94 oC

Melting

94 oC

AnnealingPrimers

50 oC

Extension

72 oCT

empe

ratu

re

100

0

50

Time

30x

5’3’

3’5’

3’5’

5’

5’3’5’

3’5’

5’

5’

5’

5’3’

3’5’

3’5’

5’3’

5’3’

5’

PCR

Fragments of defined length

5’

5’

5’

5’

5’

5’

5’

5’

PCR

Strands that are too long double in size whereas strands that are just right

increase exponentially

0Cycles

Number1

3

8

2

4

1

2

4

16

5

32

6

64

DNA Amplification

Theoretically, the number of DNA fragment copies obtained can be calculated

Yield = 2n y

Where y is the initial number of DNA copies and n is the number of thermal cycles

= 4,294,967,296,000

If you start with 1000 copies, how many copies are made in 32 cycles?

2n x y

= 232 x 1000

= 4,294,967,296 x 1000

PCR Yield

Thermal Cyclers

Challenges Fidelity of the Reaction

• Taq DNA polymerase lacks the proof-reading activity present in other polymerases

• Taq makes 1 error per 1 104 nucleotides (remember, 1 per 1 109 nucleotides in vivo)

• Thus, a 400 base pair target will contain an error in 33% of molecules after 20 cycles

• Error distribution will be random

• Does not matter if PCR product is for sequencing or to be cut with restriction enzymes

• Does matter if you want to clone the product (use proof-reading thermostable enzyme)

Optimising the PCR Reaction

• The amount of template and polymerase

• Annealing temperature of the primers and their design

• The concentration of Mg2+ in the reaction

• The extension time and temperature

• The denaturing and annealing times

The use of PCR

• PCR is a DNA ‘amplification’ method, many copies of any DNA template can synthesised

• One starting DNA template can be amplified in to an infinite number of copies

• “Amplified” fragments of DNA can be sequenced, cloned, probed or sized using electrophoresis

• Defective genes can be amplified to diagnose illnesses

• Genes from pathogens can be amplified to identify them (e.g. HIV)

• Amplified fragments can act as genetic fingerprints using restriction enzymes (nucleases cut, shorten or degrade DNA, Ligases join

DNA, polymerases make DNA copies)

PCR Practical Example Genotyping Loa mice

+/+ Loa/+

T-to-A transversion in the Dnchc1 gene that results in residue 580 changing from phenylalanine (TTC) to tyrosine (TAC)

PCR Practical Example Genotyping Loa mice

DNA prep from mouse tail Biopsies

• Cut 0.8-1.0 cm of mice tail (or equivalent mass of other parts), divide into small pieces and transfer into Eppendorf tube

• Add 300l of Lysis buffer and 3l of proteinase K. Incubate tubes at 55C overnight (lysis buffer- 100mM Tris-HCl pH8.0, 5mM EDTA, 0.2% SDS, 200mM NaCl, Proteinase K stock is 20mg/ml New England Biolabs) and leave to digest overnight

• Vortex each tube well. Spin 10-15 min to pellet hair etc…

• Pour supernatant into empty Eppendorf tube

• Dilute 4l in 200l H2O and use 2l of this in 20l PCR reactions for genotyping

PCR Practical Example Genotyping Loa mice

PCR Mix per tube

HotStar Taq master mix (Qiagen) 10ulMDN-Int7-F (10 uM) 2 ulMDN2064-R (10 uM) 2 ulH2O 4 ul

-----18 ul

+ 2ul of 1:50 diluted DNA template

Thermocycler

1) 95C 15min2) 95C 30S3) 62C 30S4) 72C 1min5) Go to 2, 35 times6) 72C 10min7) 16C Hold

PCR Practical Example Genotyping Loa mice

TGCTGCTGAGCTGCGTCCTAGTGCTGTGTGCTCTCCTGTTTTCATTCCCTCTTCACATTCATTAGTTCTTTCCTTTAAGTATACACACACACACACACACACACACAGTAAAGACAGAAGTCTGCAGGGAGATCCTTATAGTGTGCTCATGGCTGAATTGTGATGATAGAGTCCTAAAGGCCTAGAAGTCAGCATTGATGCAAGAATCCTGTGCTGTGCCTGTGACAGAAAAACGTCATTTGCAGCTATGTTTTGTTCCAAACCTTTTGTTTTAGGTCACAGCAGTCGCACAACAGAACCAAGGAGAAGCACCTGAACCCCAAGACATGAAAGTGGCCGAGGTGCTCTTTGATGCTGCCGACGCCAACGCCATTGAGGAGGTGAACCTGGCCTACGAGAATGTCAAGGAAGTCGATGGTCTGGATGTTTCCAAAGAAGGGACGGAAGCCTGGGAGGCCGCGATGAAGAGATACGATGAGAGGATCGACCGTGTGGAGACCCGCATCACCGCCCGCCTCCGAGATCAGCTCGGCACGGCCAAGAATGCCAATGAGATGTTCAGGATTTTCTCCAGGTTCAATGCACTGTTCGTCCGCCCACACATCCGAGGGGCCATTCGTGAATACCAGACCCAGCTGATCCAACGTGTGAAAGATGACATCGAATCTCTGCACGACAAGTTCAAGGTCCAGTACCCGCAAAGCCAAGCTTGTAAAATGA

Forward Primer

Reverse Primer

T-to-A change

Amplified fragment is 696 bp long

PCR Practical Example Genotyping Loa mice

PCR Product

Use agarose gels (typically 2% w/v)

Incorporate ethidium bromide or other DNA dye

PCR Product

PCR Practical Example Genotyping Loa mice

PCR Product Digestion

Digestion with RsaI (GT|AC) at 37°C for 2 hrs

672bp537bp 135bp 24bp

Wt + +

Loa/Loa

+ + +

+/Loa + + + +

PCR Practical Example Genotyping Loa mice

PCR Product Digestion

wt Loa/wt Loa/Loa

672 bp

537 bp

24 bp

135 bp

Further PCR

• Quantitative (Real-Time) PCR (Q-PCR)

• Reverse Transcription PCR (RT-PCR)

• Multiplex-PCR

• Helicase Dependant Amplification (HAD)

END

Examples