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 • Mitochondrial DNA – Part of cell nucleus – Part of cytoplasm – Found in sweat, skin, – Used mostly for bones and hair samples blood, tissue, dandruff, mucus/saliva, semen, – Two regions are highly variable vaginal or rectal cells, (Hypervariable) hair, and urine – One cell contains – Comes from 23 hundreds of chromosome pairs, mitochondria but only contributed by both the one set of nDNA mother and the father – Time consuming and costly – Less reliable in court DNA Types • Male Y-DNA – A part of nuclear DNA, found on the Y- chromosome – 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 Provides direction for further quantity of sample test Time consuming Time efficient (under 2 hours) No gender determination Provides gender Sample size needed determination depends on number of Minimal sample needed tests to be performed Amplification is slightly less Amplification very effective effective Linear results (R2=1) 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?