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Flashcards in mtDNA analysis Deck (18)

Why is mtDNA better conserved in degraded samples?

Because it is circular and does not degrade thta fast. Also, there are a lot more copies of it than of nuclear DNA


Some characteristics of mtDNA

  • maternally inherited
  • no recombination
  • haploid
  • lack of efficient DNA repair system - more variation
  • evidentiary value much lower than for nuclear DNA
  • no introns


How many genes does the mtDNA carry?

37 genes in total:

  • 22 tRNA genes
  • 2 rRNA genes
  • 13 proteins which are subunits of
    • NADH dehydrogenase
    • cytochrome oxidase
    • ATP synthase
    • chytochrome b


Describe the steps of Sanger sequencing

  1. Standard PCR - check for positive products in agarose gel
  2. Clean up of PCR products - remove remaining nucleotides and primers
  3. Sequencing - only one primer used for amplification and pyrosequencing
  4. Clean up and percipitation of sequencing PCR products - remaining primers and dyes must be removed
  5. Sequencing instrument is loaded


What do you need to do to compare mtDNA sequences?

allign them


What are the thumb rules for heteroplasmy or a mixed sample?

  • Whole HV1 and 2 have only one site of mixture - heteroplasmy
  • two or more: contamination or mixture


What is heteroplasmy?

When an individual has more than one mtDNA type

Cells may contain one type, a mixture of types, or the second type

It is often associated with diseases


Where do you use controls?

  • in DNA extraction: you have 1 negative control
  • in DNA amplification: you have 1 negative control and the negative control from the DNA extraction


What does quality assurance look like in a case?

  • one case and only one item from that case investigated at a laborartory at a time
  • items of evidence extracted and amplified prior to known reference samples or in a physically separated room
  • PCR repeated independently at least twice


How many mtDNA profiles are found in EMPOP?

34 617


What is the gold standard technique for mtDNA analysis?

 Sanger sequencing


Describe the primer extension protocol

  1. target amplification
  2. purify PCR products/eliminate primers and dNTPs
  3. SNaPshot reaction kit (with ddNTPs)
  4. [F]ddNTP cleanup
  5. electrophoresis/analysis


Name some characteristics of single base extension

  • primers with different lenghts (T-tail)
  • locus specific


Name two other techniques used in SNP detection

  • ASO: allele-specific oligonucleotide: probes that include the SNP are hybridised with the target DNA, only perfect matches will hybridise
  • SSO: sequence-specific oligonucleotides: membrane typing; biotin is added to the PCR product, it binds to a probe fixed on a membrane, washing step to remove all mismatches, then you add horse raddish peroxidase and TMB --> colouring


What are the four enzymes used in pyrosequencing?

  • polymerase
  • sulfurylase
  • luciferase
  • apyrase


Describe the pyrosequencing technique

  • dNTPs added one at a time
  • Incorporation by polymerase
  • PPi released --> converted to ATP by ATP-sulfurylase
  • Luciferase uses ATP to generate light
  • dNTPs and ATP degraded by apyrase - new cycle


The light in pyrosequencing is proportional to

  • released PPi
  • incorporated nucleotides


Name some potential problems that can occur when mtDNA profiling

  • DNA contamination - during extraction?
  • PCR product/DNA contamination - safeguards?
  • sample mix-ups?
  • technical problems
    • cross-hybridisation - strip typing
    • mispriming - sequencing
  • mixed samples - interpretation