TECHNIQUES OF DNA ANALYSIS

10%

who proposed first double helix structure of DNA

Francis Crick

James Watson

Rosalind Franklin

colin pitchfork case

2 15 year girls were r*ped and murdered in Leicestershire England in 1983-86

Alec Jefferys from Leicestershire University developed DNA testing

Performed DNA testing on over 4500 young men

Pitchfork initially avoided test by having coworker take it for him

1987 - Pitchfork was arrested

1988 first criminal caught with DNA evidence

CODIS (the compiled convicted offender database in the USA)

combined DNA index system

DNA first introduced in the USA in…

1986 and to the FBI in 1988

percentage of DNA which codes for proteins?

3%

percentage of noncoding?

97%

satellite DNA

This 1% DNA sequence is present as small stretch of repeated sequences

Microsatellites/short tandem repeats (STRs)

2-7 bps in each tandem repeat

minisatellites/ variable tandem repeats

a given sequence is repeated 5 times in the haploid DNA in sperm and 12 times in the haploid DNA in the egg

child will have genotype/phenotype 5/12

heterozygosity

individual has 2 different copies of allele (5/12)

Homozygosity

when an individual has same number of copies at both loci (12/12)

Restriction fragment length polymorphism (RFLP)

technique used by Alec Jeffreys

no longer used

Modern Technique for DNA typing

short tandem repeat (STR) analysis

sources of DNA collection

blood

semen

bone

hair

dried skin

nucleases

enzymes responsible for most environmental breakdown of DNA

found nearly everywhere

PCR

polymerase chain reaction

Denaturation

breaks DNA backbone by heating 95C

Annealing

primers bond through complementary base paring

50 DNA can bond together but the primers prevent this

Extention

72 activates DNA polymerase and extends primers by adding complimentary bases

until reaches primers

PCR is an

Exponential process

what are the copies of PCR DNA called

amplicons

short tandem repeats

vary in the number of repeats each person inherits from their parents

mid 1990s 13 core loci were chosen as the standard loci to be typed and used in CODIS

capillary electrophoresis (fast and automated)

DNA is detected using laser-induced fluorescence (LIF) where amplicons are labeled with one of 4 dyes

amplicons are separated in the capillary based on their relative size and charges

at end the column amplicons pass a laser causing the dyes to glow generating a colour-coded signal that is measured

peaks noted

each sample yields 15 phenotypes

mitochondrial DNA

mtDNA is inherited from the mother

all members of the linage will share the same mtDNA

mtDNA mutates at a higher rate than nuclear DNA

hypervariable regions

HVI, HVII and HVIII used for analysis

y-chromosome short tandem repeats

used to distinguish male DNA from female DNA

a set of STR markers associated with just Y-chromosomes has been developed

can find if a male has contributed to mixed sample