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					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?

				
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