mtDNA analysis

Question 1

Why is mtDNA better conserved in degraded samples?

Answer 1

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

Question 2

Some characteristics of mtDNA

Answer 2

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

Question 3

How many genes does the mtDNA carry?

Answer 3

37 genes in total:

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

Question 4

Describe the steps of Sanger sequencing

Answer 4

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

Question 5

What do you need to do to compare mtDNA sequences?

Answer 5

allign them

Question 6

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

Answer 6

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

Question 7

What is heteroplasmy?

Answer 7

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

Question 8

Where do you use controls?

Answer 8

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

Question 9

What does quality assurance look like in a case?

Answer 9

• 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

Question 10

How many mtDNA profiles are found in EMPOP?

Answer 10

34 617

Question 11

What is the gold standard technique for mtDNA analysis?

Answer 11

Sanger sequencing

Question 12

Describe the primer extension protocol

Answer 12

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

Question 13

Name some characteristics of single base extension

Answer 13

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

Question 14

Name two other techniques used in SNP detection

Answer 14

• 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

Question 15

What are the four enzymes used in pyrosequencing?

Answer 15

• polymerase
• sulfurylase
• luciferase
• apyrase

Question 16

Describe the pyrosequencing technique

Answer 16

• 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

Question 17

The light in pyrosequencing is proportional to

Answer 17

• released PPi
• incorporated nucleotides

Question 18

Name some potential problems that can occur when mtDNA profiling

Answer 18

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