DNA Profiling II

Allele frequencies

• To calculate the significance of a match the frequency of an allele must be calculated

• Number allele occurs / Total number of possible alleles = frequency of that allele

If 20 of a given allele is observed in a population group of 100, the frequency would be…

20 / 200 = 0.1

Hardy-Weinberg equations

PP (homozygotic) = p²

QQ (homozygotic) = q²

PQ (heterozygotic) = 2pq

• Find allele frequency on the allelic frequency table

• Multiply all the results of each STR together

• Then do 1 / answer

Example of Hardy-Weinberg

Example 2

D2 – 17, 19

Heterozygotic, so 2pq

2 x 17 x 19

Example 3

THO1 – 9.3, 9.3

Homozygotic, so p2

9.3 x 9.3

The National DNA Database (NDNAD)

• Held by the Home Office

• Criminal justice forces feed into the database

• Buccal scrape kit (a cheek swab) is how DNA data is collected

• Current criteria for sampling is all arrestable offences

• Samples are kept in the UK, but in other countries this can differ

Missing Persons DNA Database (MPDD)

• 2000 profiles held on here from people who have disappeared or family members who have given their profiles for matching

• Not held on the NDNAD

• Each year these produce ~15 matches

Vulnerable Persons DNA Database (VPDD)

• Anyone thought to be at high risk can request their profile is added

• Not held on NDNAD

• 6200 profiles held

• 1-2 matches per year

Police Elimination Database (PED)

• All serving police profiles held for elimination from crime scene samples

• Held for 12 months after they leave

Removal of samples

• DNA profiles and fingerprints of anyone convicted of a recordable offence will be stored permanently

• Those obtained on arrest even with no conviction will be stored for 6 years

• Renewable on new arrests

Mass screens

• Involves major crime, murder, abduction or rate

• Where police believe the offender is local

• Where the population of potential offenders can be targeted

SNP analysis advantage

• Good if the DNA is heavily degraded and contains short fragment sizes of 40-50 bp

• SNP detection is highly suited to high throughput automated methods

SNP analysis disadvantage

• Need to do more SNPs to get same information as STR typing

• Require 50 – 70 SNPs to match STR analysis

SNP classifications

• Transition

• Transversions

• Synonymous

• Non-synonymous

Transition

Same base is retained e.g. purine to purine

Transversions

Base change e.g. pyrimidine to purine

Synonymous

Mutation does not change the amino acid

Non-synonymous

Mutation does change amino acid

Genetic markers

• Large numbers

• Stable

• Spread out uniformly across genome

Genetic markers uses

• Complex diseases

• Pharmacogenomics

• Genetic markers

• Forensics

• Population, migration genetics

Genetic markers forensic uses

• Mitochondrial DNA sequences

• Y chromosome genetic markers in paternity cases or sexual assault

• Degraded DNA samples

SNPs for physical appearance

• Tells you above physical appearance and height

• Pigmentation can tell location on body, age and gender

• Geographic ancestry

Detecting SNPs

1. Restriction digests

2. Molecular beacons

3. Multiplex PCR

4. Sequencing

Restriction digests

• Make an EcoRI site GAATTC

• Can check if they have SNP

• If there is an SNP, it works vs if it doesn’t, there is no SNP

Molecular beacons

• Hairpin-shaped fluorescent hybridisation probes

• The ends of the oligonucleotide are designed to be complementary to each other and can form a stem structure

• Intervening loop is complementary to a sequence within the amplified product

• In solution, the unhybridised probe adopts a hairpin structure

• If the molecular beacon is bound to its complementary target, the fluorophore and quencher are far enough apart that there is no quenching and the molecular become fluoresces

• This can be detected in a real-life PCR machine

Molecular beacon results

The wild type is red vs SNP is green

• Homozygous WT – only red

• Homozygous SNP – green

• Heterozygous – green + red

Multiplex PCR

• Amplify mitochondrial PCR

• Design set of forward and reverse primers which will produce different size amplicons covering different SNP sites

• Protocol – amplify DNA with multiplex primers (10 SNPs)

• Primer extends using SNaPshot – analyse sequencing on capillary gel DNA detection system

Sequencing

• Next-generation throughput sequencing

• Sanger-based dideoxy sequencing using fluorescent dye labelled terminators

• Each nucleotide base has a different coloured dye which allows sequences to be determined from a single lane on a capillary gel by a scanning laser

• Analysis on the Beckman CEQ 800

Analysis profiling

Use SNP-based profiling to produce a phenotypic description rather than DNA fingerprint

DNAWitness

• This gives percentage of European, East Asian, Native American and Sub-Saharan African from DNA using 176 SNPs

• Ratio of each for an individual is called the Bio-Geographical Ancestry (BGA) profile

• Can use profile to give general characteristics of a suspect or victim without prior knowledge of the person