Invention of PCR Kary Mullis

Mile marker 46.58 in April of 1983

Pulled off the road and outlined a way to conduct DNA replication in a tube

Worked for Cetus, which was purchased by Chiron and is now part of Novartis

What is PCR? Polymerase chain reaction or PCR

Simplified version of bacterial DNA replication

Copies a specific sequence of DNA

Target sequence

PCR products

Amplicons

Target is determined by the primers

Short sequences of single-strand DNA

To amplify a defined segment, two are required

Annealing is when the primers bind to the target sequence

The two primers are referred to as forward and reverse primers

Challenges of DNA Replication in a Tube Normal replication uses enzymes

that are heat sensitive.

Using heat to denature the double strands of DNA, would damage the enzymes.

Heat-stable DNA polymerases were needed.

Thermus aquaticus was the bacteria from which the first heat-stable DNA polymerase was isolated and the enzyme was named Taq polymerase

Materials of PCRtarget DNATaq DNA polymerase2 Primers

~20 nucleotides in length Forward and reverse

the four DNTP’S AdenineThymineCytosineGuanine

cofactor MgCl2.

Primers: Two different primers are used because:

The backbones run in opposite directions.

The beginning and the end of the target sequence need to be marked.

Taq Polymerase must have a beginning point to build the rest of the DNA onto.

DNTPs: DNTPs are the

nucleotides that the new DNA is built from.

Locate the Target Sequence

Scientists determine which GENE they are interested in replicating.

Determine where primers would bond upstream and downstream of at each end of the gene.

Thermal Cyclers Initially water baths were

used, but that was very labor intensive.

Thermal cyclers are electronically controlled heat blocks that can quickly change from one temperature to another.

They are programmable and are in many different configurations depending upon their purpose.

Steps of PCR: Denaturing:

Separation of the backbones by heating.

94ᴼ C for 60 sec.

Annealing:

Bonding of the Primers.

50ᴼ - 65ᴼ C for 60 sec.

Extension:

Elongation or building of the rest of the backbone.

72ᴼ C for 2 min.

Cycling temperatures and times

The amount of time that each step is held depends on the length of PCR product and the amount of time it would take to amplify that length. Shorter lengths require shorter times

Generally only the annealing temperature is adjusted

To ensure all DNA is denatured

Denature DNA

Bind primers Extend primers

Repeat this 40 times

Finish any products

Hold until ready to remove from thermal cycler

Steps in 1 cycle of PCR:

Step 1 - Denaturing

• 60 seconds

• @ 94°C

• Backbones separate.

Step 2 - Annealing

• 60 seconds

• @ 60°C

• Forward and Reverse Primers

bond to the template.

Taq Polymerase Binds to the primers

Step 3 - Extension

• 2 minutes

• @ 72°C

• Taq Polymerase uses DNTP’s to build

the rest of the new backbones.

Multiplex PCR

Over 10 Markers Can Be Copied at Once

Sensitivities to levels less than 1 ng of DNA

Ability to Handle Mixtures and Degraded Samples

Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges

Available Kits for STR Analysis

Kits make it easy for labs to just add DNA samples to a pre-made mix

13 CODIS core loci Profiler Plus and COfiler (PE Applied Biosystems)

PowerPlex 1.1 and 2.1 (Promega Corporation)

Increased power of discrimination CTT (1994): 1 in 410

SGM Plus™ (1999): 1 in 3 trillion

PowerPlex ™ 16 (2000): 1 in 2 x 1017

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DNA Size (base pairs)

Results obtained in less than 5

hours with a spot of blood the

size of a pinhead

probability of a random

match: ~1 in 3 trillion

Human Identity Testing with Multiplex STRs

Simultaneous Analysis of 10 STRs and Gender ID

AmpFlSTR® SGM Plus™ kit

DNA Sequencing Modern sequencing techniques use

PCR to amplify the sequence to be determined

A mixture of terminating and non-terminating nucleotides are used in the reaction

Each terminating nucleotide has a different color dye added to it, a dideoxynucleotide triphosphate (ddNTP). Once the growing chain terminates, the nucleotide will have a unique color associated with it

DNA Sequencing Since the primer is

known, each successive terminated fragment should be one nucleotide longer

Automated sequencers read the colors as they electrophorese past a detector

PCR in forensics

Short tandem repeats (STR) analysis

Short tandem repeats are amplified and used as a method of identification

They have repetitive regions from 2-6 bp

They are inherited from parents

They display lots of variety

Other Applications of PCR PCR in forensics

– Allele frequencies

• Vary between different ethic groups

– Combined DNA Index System (CODIS)

• Indexes 13 different STR loci

• Maintains national sample database FBI uses to statistically calculate random match probability

Gel Results Compare crime scene and suspects to allele ladder

Determine the genotype by recording the allele combinations

Determine if each suspect can be removed as a candidate from crime scene

Allele Ladder