PCR and Forensics

YouTube - CSI Intro

What we’ll be doing

• Lesson 1 DNA chemistry

• Lesson 2 Theory of PCR

• Lesson 3 Prac 330: PCR using supplied template (set up and run PCRs)

• Lesson 4 Run gel of PCRs from Lesson 3, Prac 225 do restriction digests.

• Lesson 5 Run restriction digests on gels and produce DNA fingerprints

Lesson 1: The chemistry of DNA

In a nutshell, PCR:

• Produces enough target DNA that it can be analysed, either on a gel or in a machine which measures fluorescence.

• DNA (deoxyribonucleic acid) is the chemical which carries the genetic code of life on this planet.

YouTube - Polymerase Chain Reaction

Pratt Ch. 3. page 54, 55

DNA bases

page 55 Nucleosides.

The bases are linked to a

five-carbon sugar to form nucleosides.

In RNA, the sugar is ribose.

In DNA, the sugar is 2’ deoxyribose.

page 56 The structure of DNA.

The structure of DNA:

The linkage between the

nucleotides

is called phosphodiester bond.

Linked nucleotides form a polymer

in which the the backbones are

the phosphate-sugar groups and

the bases project out.

One

strand

The opposing strands of DNA pair anti-parallel

5’

3’ 5’

3’

Exercise

• Draw a 3 base section of DNA indicating the 5’ and 3’ regions

• Look at the next slide and explain, using your knowledge of electronegativity and molecular geometry, explain how/why the hydrogen bonds shown exist

• Here’s a really tough one: hydrogen bonding doesn’t have an effect on double strand stability, why not?

• The pKA of the phosphoryl groups is about 3. Explain why DNA is negatively charged at pH 7.0

page 57 DNA base pairs.

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DNA contains 2 strands (double

helix) as their bases pair

through hydrogen bonding.

Which is

stronger??

How is DNA measured?

• Size of DNA is measured in base-pairs (bp) or, for example, Kilobase pairs (1000bp=1kb).

• Most DNA in cells is thousands to millions of bp

Genome Sizes of Model SystemsModel system Size (Million Bases) Genes

Escherichia coli (Bacterium)  4.6 3,000

S. cerevisiae (Yeast) 15

Neurospora crassa (Fungus) 39.9 10,000

What is the mass of one human genome? Assume:

• Genome is 109bp

• The average mass of a nucleotide residue is 300g/mole

DNA replication issues

• DNA polymerase must have a primer (a bit of DNA or RNA on which to elongate the DNA strand)

• The raw material used to construct DNA is dNTPs

• DNA polymerase can only synthesise 5’ 3’

Draw a diagram illustrating how DNA is synthesised.

Lesson 2: The theory of PCR

POLYMERASE CHAIN REACTION

Polymerase chain reaction (PCR) uses thermostableDNA polymerase and specific oligonucleotide primersto amplify genes.

The first thermostable DNA polymerase was Taqpolymerase from the bacterium Thermus aquaticuswhich was isolated from hot springs.

Figure 3.08 A DNA melting curve.

Figure 3.09 Renaturation of DNA.

The rate of renaturation depends on the

length of

the DNA fragment Short fragment anneal

faster

than long fragments.

POLYMERASE CHAIN REACTION

Denature DNA sampleto separate DNA strands

(94oC, 5 min)

Hybridise primersto DNA strands(30-65oC, 30 s)

Taq polymerasesynthesises new

DNA strands(65-75oC, 2-5 min)

Denature toseparate newDNA strands(94oC, 30 s)

20-30 cycles

POLYMERASE CHAIN REACTION

5’3’

3’5’

Denature DNA

3’

5’ 3’

5’

Anneal specific primers

3’

5’ 3’

5’

Target DNA

POLYMERASE CHAIN REACTION

5’3’

3’5’

Extend primers withTaq polymerase

3’

5’ 3’

5’

5’3’

3’5’

Denature DNA andanneal specific

primers

Extend primers withTaq polymerase

Repeat cycles

3’

5’ 3’

5’

5’ 3’

5’3’

3’

5’ 3’

5’

5’ 3’

5’3’

Size of PCR fragment

POLYMERASE CHAIN REACTION

Cycle number123456789

101112131415

Target DNA01248

163264

128256512

1024204840968192

Cycle number161718192021222324252627282930

Target DNA163843276865536

131072262144524288

1048576209715241943048388608

167772163354443267108864

134217728268435456

The PCR Song and a myth• YouTube - PCR• YouTube - The PCR Song• YouTube - CSI PCR in 60 seconds: BS

There was a time when to amplify DNA,You had to grow tons and tons of tiny cells.Then along came a guy named Dr. Kary Mullis,Said you can amplify in vitro just as well.Just mix your template with a buffer and some primers,Nucleotides and polymerases, too.Denaturing, annealing, and extending.Well it’s amazing what heating and cooling and heating will do.PCR, when you need to detect mutations.PCR, when you need to recombine.PCR, when you need to find out who the daddy is.PCR, when you need to solve a crime.

POLYMERASE CHAIN REACTION

PCR is now used routinely for:

• cloning genes• DNA sequence analysis• site directed mutagenesis• generating DNA probes• random PCR to differentiate individuals• cloning flanking DNA• Producing DNA from unculturable

microorganisms

Cancer detection

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Typical PCR reaction mixtureCOMPONENT VOLUME FINAL

CONCENTRATION

1. autoclaved ultra-filtered water (pH 7.0)

20.7µL -

2. 10x PCR Buffer* 2.5µL

3. dNTPs mix (25 mM each nucleotide)

0.2µL

4. primer mix (25 pmoles/µL each primer)

0.4µL

5. Taq DNA polymerase (native enzyme)

0.2µL 1 Unit/25 µL

6. genomic DNA template (100 ng/µL)

1.0µL

* The PCR buffer used was made after the recommendations of the manufacturer/vendor (Perkin Elmer Cetus). The 10x PCR buffer contains: 500 mM KCl; 100 mM Tris-HCl (pH 8.3); 15 mM MgCl2 (the final concentrations of these ingredients in the PCR mix are: 50 mM KCl; 10 mM Tris-

HCl; 1.5 mM MgCl2).

It is useful to prepare a larger volume of this buffer (10-15ml), aliquot it and store the vials at -20 C for years.

Calculate the final concentrations of the components in the table below.

Things Are a Little Different Now...

PCR Robot from the early 80’s

PCR is a little trendier these days...

Now try the PCR/bioinformatics exercise…