Multiplex PCR for quantitation of DNA

Biochemistry Article Presentation

Mandy Eves Lynette Nolt

March 30, 2005

Learning Objectives
Know the three types of DNA and their importance in forensic applications Understand the process of the Polymerase Chain Reaction (PCR)

Explain the advantages/disadvantages of multiplex assays in quantitation of DNA

DNA Types
Nuclear DNA
Part of cell nucleus Found in sweat, skin, blood, tissue, dandruff, mucus/saliva, semen, vaginal or rectal cells, hair, and urine Comes from 23 chromosome pairs, contributed by both the mother and the father

Mitochondrial DNA
Part of cytoplasm Used mostly for bones and hair samples Two regions are highly variable (Hypervariable) One cell contains hundreds of mitochondria but only one set of nDNA Time consuming and costly Less reliable in court

DNA Types
Male Y-DNA
A part of nuclear DNA, found on the Ychromosome Used to trace origin of modern man Gender determination in forensics
Also used in the Olympics for gender verification of athletes

Unfamiliar Terms
Polymorphic short tandem repeats (STRs)
The defined region of DNA with multiple copies of base pairs which vary among the population

Restriction fragment length polymorphisms (RFLP)

Different lengths of chromosomes due to genetic differences caused by enzymes

TaqMan-MGB probes
A molecule of single stranded DNA that fluoresces

Unfamiliar Terms
Primer
Short oligonucleotides that are precisely complementary to the sequence at the 3' end of each strand of the DNA you wish to amplify.

Cross-Amplification
The interference of another species DNA sequence in PCR

Polymerase Chain Reaction (PCR)

The method for amplifying DNA sequences

Research Purpose
To create an improved triplex method of DNA quantitation
1. Human specific 2. Target specific 3. Multiplex compatible

To compare with already established duplex method Main use in forensic genomics

Methods: Primer and Probe

What is a primer? Primer was specific to the chosen target sequence on DNA
nDNA sequence: 7-bp duplicated region mtDNA sequence: human specific region Male Y-DNA: 167-bp fragment

What is a probe?
TaqMan-MGB probe fluoresces under UV light

Method: PCR Amplification

Strand Separation
Heating

Hybridization of primers
Cooled

DNA Synthesis
Heated Taq DNA polymerase

PCR Amplification is continued by cycles of heating and cooling

PCR animation

Method: PCR Amplification

Only need to know flanking sequences Target can be much larger than the primer Primers do not have to be perfectly matched to target sequence
Good or Bad?

It is highly specific PCR is extremely sensitive

Only single molecule of DNA necessary

Method: DNA Samples

Humans
HeLa cell line Human-rodent somatic cell

Animals
Pygmy chimp, common chimp, gorilla, dog, cat, rabbit, horse, cow, sheep, pig, deer, rat, mouse, chicken Purchased Coriell Cell Repositories Obtained by tissue and blood extraction

Method: Data Analysis

Used Excel to construct standard curves
duplex & triplex

Pair wise t tests

To determine statistically similarities

Experimental Method
Bought or procured the target sequences of each species Performed PCR Ran the samples on an agarose gel Stained with ethidium bromide Detected using fluorescence. Graphed data and analyzed using excel.

Results
ABI Prism 7000 SDS software was used to measure the fluorescence of ethidium bromide Threshold Cycle: the number of PCR cycles necessary for the DNA fluorescence to surpass the baseline fluorescence

Results: Duplex
Includes nDNA and mtDNA Quantitation range 100-0.001 ng Human DNA can be detected in up to 50% contamination

Cross-Amplification: Duplex
Before 32 PCR cycles
nDNA: negligible for all 14 species mtDNA: absent for all 14 species

Results: Triplex
Includes nDNA, mtDNA and male Y-DNA Quantitation range 100-0.10 ng

Cross-Amplification: Triplex
Before 39 PCR cycles
nDNA: negligible for 11 of 14 species mtDNA: absent Male Y-DNA: some with 3 primates of 14 species

Considered human specific overall

Discussion
Duplex

Triplex

Choose tests based on quantity of sample Time consuming No gender determination Sample size needed depends on number of tests to be performed Amplification very effective Linear results (R2=1)

Provides direction for further test Time efficient (under 2 hours) Provides gender determination Minimal sample needed Amplification is slightly less effective Less linear results (R2=.9958)

Discussion: When should each method be used?

A duplex method should be used when:
A sample is less than 0.1 ng A suspect is known to be a female Dealing with a sexual assault case

A triplex method should be used if:

Gender is undetermined Contamination of the DNA sample is suspected The sample is degraded (smaller target sequence during PCR)

Discussion Questions
How is PCR useful in forensic science? Explain one of the many advantages of PCR, what makes PCR special? In what ways will this study help aid forensic science? Name one type of case that a duplex method should be used over the new triplex method. Give an advantage of the triplex method. Which type of DNA is most often used in forensic cases?