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Forensic Genetics and Medicine > Short tandem repeats in forensic genetics > Flashcards

Short tandem repeats in forensic genetics Flashcards

1
Q

The history of DNA testing

A
  • RFLP (1985-1990)
  • DQ-alpha (1990-1995)
  • automated STR (1995-)
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2
Q

what are STRs

A
  • microsatellites
  • core repeat motive of 1 to 6 bp
  • <350 bp long (alleles)
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3
Q

Where are STRs used?

A
  • human identification
  • paternity testing
  • missing perso cases
  • violent crimes
  • mass disasters
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4
Q

How are STRs classified? (4)

A
  • simple repeat
  • simple repeat with non-consensus allele
  • compound repeat - composed of several elements
  • complex repeat
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5
Q

Name characteristics that STRs should possess in order to be used in forensics?

A
  • tetra or penta repeats
  • discrete and distinguishable alleles
  • amplification of locus should be robust
  • a high power of discrimination
  • absence of genetic linkage with other loci
  • low levels of artifact formation during the amplification
  • the ability to be amplified as part of a multiplex PCR
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6
Q

Describe the nomenclature of a STR

A

D3S1358

D = DNA
3 = number of chromosome
S = unique sequence
1358 = particular sequence
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7
Q

What is the amelogenin gene?

A
  • Encodes a protein in tooth enamel
  • not an STR, two alleles
  • present on both X and Y chromosome
  • 6bp longer on Y chromosome
  • used for sex determination (but not an absolute indicator)
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8
Q

Describe the workflow of DNA profiling

A
  • DNA extraction
  • preparation of PCR reactions
  • PCR
  • gel electrophoresis
  • Sanger sequencing
  • Capillary electrophoresis
  • analysis with software
  • profile
  • profile evaluation
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9
Q

How does capillary electrophoresis work?

A
  • glass tube fille with polymer solution
  • DNA moves from negative to positive pole (to anode)
  • argon laser through capillary, hits label on PCR product and excites fluorophores –> emission of fluorescent light
  • light passes through filter (removes background noise)
  • light passes through charged coupled device (CCD) camera detecting wavelength
  • information to computer –> electropherogram
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10
Q

What is an electropherogram?

A

A plot of results from an analysis done by electrophoresis - can be used to determine DNA sequence genotypes

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11
Q

Name characteristics of fluorescent based detection assay

A
  • dye binds to 5’ end of primer
  • detected real-time during electrophoresis
  • up to five different dyes - overlap of loci possible
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12
Q

What is the internal lane size standard?

A
  • fragments of DNA of known length, fluorescently labeled
  • size in bp
  • allows for comparison and ensures consistency
  • comes with the kit
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13
Q

What is the allelic ladder?

A
  • included in kit
  • artificial mixture of all human alleles
  • reference size for each allele
  • migration of PCR products varies
    • temperature
    • electropheric conditions
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14
Q

Name some complications that can occur with STR analysis

(Also including the profile itself)

A
  • stutter peaks
  • split peaks
  • pull-ups
  • overloaded profiles
  • mixed samples
  • DNA degradation/low template DNA
  • PCR inhibition
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15
Q

What are stutter peaks?

A
  • one repeat unit smaller or larger than the true allele
  • caused by strand slippage during extension
  • do not interfere with interpretation of profile
  • seens as smaller peak next to bigger
  • threshold limits: <8-15% of main peak
  • shorter STRs more prone to stutter
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16
Q

What are split peaks?

A
  • Taq-polymerase is a terminal transferase - template dependent vs. non-template dependent
    • I.e. sometimes it puts a random nucleotide to the extended strand that does not correspond to the template. in 85% A is added
  • caused by:
    • taq polymerase suboptimal activity
    • too much DNA in the PCR
  • prevention: add incubation step to PCR
  • profile often still readable (only 1bp dif)
17
Q

What are pull-ups?

A
  • caused by
    • matrix file poor quality
    • over-amplification
  • peaks in profile composed of more than one colour
  • easy to recognise: smaller product of same size as allele
18
Q

What are overloaded profiles and how do you get them?

A
  • overloaded PCR –> CCD camera saturated –> peak height not reliable
  • noisy background
  • stutterings
  • split peaks
  • pull-ups
19
Q

What can occur during low template DNA typing?

A
  • low copy numer <100 pg DNA
  • LCN –> increased number of cycles in PCR
  • allele drop-in or drop-out
  • peak imbalance
  • locus drop-out
  • increased stutter
20
Q

What is allelic drop-in?

A
  • false PCR products from unkown DNA –> extra allele
  • completely random so rarely repeated
  • set up samples many times
  • at least two amplifications of same sample as routine
21
Q

What is allele drop-out?

A
  • one allele preferentially amplified
  • caused by
    • mutation in primer binding site
    • primer mismatch
    • degradation
    • initial input of DNA too low
    • allele size outside normal range goes undetected
  • heterozygous appears homozygous
  • repeat PCR twice
22
Q

How to assess peak height imbalance

A
  • peak size is proportional to amout of PCR product
  • peak high balance should be 1:1
  • cutt of value 60-70%
  • height of smaller peak divided by height of larger peak
23
Q

How does one assess mixed profiles? (5 steps)

A
  1. Identify the presence of a mixture
  2. identify the number of potential contributors
  3. estimate the relative ratio of the invidividuals contributing to the mixture
  4. consider all possible genotype combinations
  5. compare reference samples
24
Q

STRs vs SNPs

  • discrimination power
  • occurence
  • multiplex capacity
  • artifacts
  • amount DNA needed
  • mixed sample analysis
  • phenotypic information
A
  • discrimination power: STRs have the higher one
  • occurance:
    • STR: every 15kb
    • SNPs: every 500bp
  • multiplex capacity:
    • STR: approx 20
    • SNP: approx 50
  • artifacts: occur only with STRs
  • amount DNA needed:
    • STR: 0,5-1ng DNA
    • SNP: 100pg DNA (degraded)
  • mixed samples:
    • STR: possible to analyise
    • SNP: difficult to analyse
  • phenotypic information: only in SNPs
25
Q

What does the likelyhood ratio indicate?

A

It indicates how many times more likely it is that a sample originates from a particular person than that originats from an unrelated person

Forensic Genetics and Medicine (19 decks)

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