DNA Profiling I

Reasons for human identity testing

• Forensic cases

• Paternity testing

• Identifying people from remains in mass disasters and human investigations

• Missing persons investigations

• Military DNA - all soldiers have DNA taken and sequenced as a ‘dog tag’

• Convicted felon DNA databases

Sources of human DNA

• Body fluids e.g. blood, semen, urine, saliva, faeces

• Tissues e.g. skin, hair, bone

• Fingerprints

• Used chewing gum

• Cigarette butt

• Weapons

• Bite marks

• Used tissue

Aim of forensic profile

• Unique profile that links a person to a crime or relative unambiguously

• DNA polymorphisms allow generation of profile

• Everyone has different numbers of the repeated sequences

Short tandem repeats (STRs)

• Known as microsatellites

• Repeating units of 1-7 bases

• 5-100 repeats at each satellite

• The core unit ranges from 2-6 bp

• Scattered through the genome

• For forensic profiling, tetra (4) and penta (5) are used

2 STR

Dinucleotide

3 STR

Trinucleotide

4 STR

Tetranucleotide

5

Pentanucleotide

6

Hexanucleotide

What four repeats are used for DNA profiling?

• TPOX - simple repeat

• THO1 - simple repeat

• D12S391 - compound repeat

• VWA - compound repeat

TPOX

• Human thyroid peroxidase gene

• Chromosome 2

• Intron 10

• Simple repeat

• Repeating unit is [AATG]

TPOX₈

[AATG][AATG][AATG][AATG][AATG][AATG][AATG][AATG]

Repeating unit is repeated 8 times

THO1

• Human tyrosine hydroxylase gene

• Chromosome 11

• Intron 1

• Simple repeats with non-consensus alleles

• THO1 repeat [AATG]

THO1_9.3

[AATG]₆ATG[AATG]₃

THO1_9.3 has one incomplete repeat with three nucleotides

D12S391

• Chromosome 12

• Compound repeats

• Two or more adjacent simple reads

• Repeating units [AGAT][AGAC]

  [AGAT] 8 to 17 in a row [AGAC] 6 to 10 in a row [AGAT] 0 to 1 in a row

D12S391₁₅

[AGAT]₈[AGAC]₆[AGAT]

VWA

• Human von Willebrand factor

• Chromosome 12

• Intron 40

• Compound repeat with non-consensus alleles

• Repeating unit [TCTA][TCTG]

VWA₁₆ and VWA_16.2

VWA₁₆ [TCTA]₂[TCTG]₄[TCTA]₁₉

VWA_16.2 [TCTA]₂[TCTG]₄TA[TCTA]₁₀

• There are two repeating units

• In certain groups of people, after the first two repeats, there is ‘TA’ and then the next set of repeats

• 16 is the full repeats and .2 denotes the two extra bases

Complex repeats

• When picking repeats for analysis, one complex repeat (always VWA) and THO1 will be used

• Mixture of simple and non-repeats

Genotype

• If the father has 7 repeats in one chromosome and 3 repeats in the other, it will be inherited by the sperm whose genotype will be 7,3

• If the mother has 4 repeats in one chromosome and 2 repeats in the other, it will be inherited by the egg whose genotype will be 4,2

• The children of this couple will then have equal likelihood to have a genotype with any combination of these repeats

PCR amplification of STR alleles

• On the graph, smaller fragments are first and then larger fragments

• Two peaks - heterozygotes (alleles different)

• One peak - homozygotes (alleles same length)

• Introduced in 1994 with 4 sites (QUAD)

SGM

• 1995

• 6 sites

• 1 in 50 million discriminating power

QUAD

• 1994

• 4 sites

• 1 in 10,000 discriminating power

SGM+

• 1999

• 10 sites

• 1 in 1000 million (1 billion) discriminating power

• In 1999, introduction of low copy number (LCN)

DNA-17

• 2014

• 17 sites

• 1 in 1000 million (1 billion) discriminating power

Discriminating power legal requirements

• Legally, can’t say there is more than 1 in a billion chance

• Unless entire population has been sequenced, can’t be certain

• This is why SGM+ and DNA-17 have the same result

Stain type

STR fragment analysis

• STR fragments can be told apart by putting fluorescent dye on the forward primer

• Attach the primer via linker onto the DNA base on the forward primer

• The succinimidyl ester reacts rapidly with amine linkers on DNA bases

• Dyes - blue, green, yellow, red

• By running it on a DNA sequencing machine, it shines the appropriate laser at the appropriate wavelength so they can tell them apart

SGM plus TM

• When D3 is amplified up, the individuals with D3 will have amplicon sizes between 80 - 135 bp, labelled blue

• D8 - STR’s amplified in that region will be between 115 - 175 bp, but this falls in the size range of D3, so they’re labelled green

• D19 sits within D3 and D8 range so it’s yellow

• A is always amplified up

• To know how big the fragments are, use a marker/ladder made of DNA fragments

A

• Amylogenic

• Sex-determining amplicon

• X - amplifies at 106 bp

• Y - amplifies at 112 bp

DNA-17 kit

• Similar to SGM+ STRs

• Has a few extra STRs

• Bigger kits need to use red so often another colour is used for the ladder

STR typing steps

1. Native acrylamide is used

2. Flush out the capillaries to clear it from previous samples

3. Load plate into machine 8 samples at a time

4. Capillary electrophoeresis

5. As it passes down, there is a window where a laser divides it into colours with a prism to then detect the colour and allow sample detection and interpretation

6. The machine and capillary site are slightly outside the box first

7. The capillaries can’t be dipped in so as it loads new samples - it moves across the first row and applies and a positive charge which moves the sample up into the capillaries (reducing contamination)

Native acrylamide

• Doesn’t denature

• Allows analysis of small differences in DNA size

Capillary electrophoersis

• Runs on a thin wire

• Filled with acrylamide

• Takes PCR samples to inject into capillary

Detection platforms for STR typing

• Beckman CEQ 8000 capillary electrophoresis

• ABI 3730xl DNA analyser (best type)

• Beckman EQ 2000XL CE-based DNA sequencing system

CEQ sample preparation

• Sample plate is a 96 well plate

• Into each well, add a sample and the buffer with the size marker

• Add 0.5µl PCR reaction into the well

• Load up the plate and run on CEQ

CEQ8000 collection software

• Samples run on one gel

• The machine run and data collection are controlled by one array

• Analysis is done with one software

• One array controls the injection of the samples and electrophoresis conditions

• it then creates a file with the data collected from the CCD

Software processes the raw data

• Matrix file - corrects for spectral overlap

• Size standard - detects the size standard in the sample’s data

• Displays data that can be used to assign allelic designations to the DNA profile

Fluorescence detection

• A dye molecule is excited by a laser

• The excited dye emits light which is then detected

• The fluorophores used in DNA detection are in visible range

• Filters detect the emission spectra of the dyes that are used

• Data is detected by a charged coupled device (CCD)

• An electron hitting this is converted into an electrical signal

• The strength of the current is proportional to the intensity of the signal

• More produced, bigger the peak

Flurorophores

Molecules capable of fluorescence

Visible range

400 - 600 nm

Spectral overlaps

• Must be able to distinguish from slight differences in different colour dyes to avoid overlap

• Must use colour corrections when sample comes off

• Matrix files correct spectral overlap

• In the raw data, it’s all mixed together as matrix correction hasn’t been applied

Matric files correct spectral overlap

• Standards are run on the detection platform

• Amount of contaminating colour is measured

• This is used to correct raw data from analysed samples

Interpretation of peaks

• Peak morphology

• Peak balance

• Peak height/area

• Artefacts

Peak morphology

Nice clear point/peak should have shoulders as it gets down to the bottom

Peak balance

Two alleles of any one loci should be balanced and if they are imbalanced, the DNA could be degraded

Peak height/area

This is worked out by the machine so they can be compared to values in the table to see whether they’re similar

Artefacts

• PCR stutter

• Non-template addition

• Allelic dropout

Stutter

• Slippage during amplification

• Mispairing during PCR reactions

• Polymerase doesn’t like repeating sequences and often misses the base

• Some peaks are smaller than the true peak in the profile

• Tetramers have less stutter than dinucleotide repeats

• Affects simple repeats

• Large alleles demonstrate a higher degree of stutter

Minimise stutter

Use complex repeats - tetra/pentra repeats and a taq with high fidelity

Non-template addition

• Taq polymerase adds an extra nucleotide to the 3’ end of the PCR product

• The nucleotide is most often an adenosine

• Taq polymerase has a terminal N (A) transferase activity - at the end of the PCR product, an adenine residue is added

• If the system doesn’t resolve to the 1 bp level, it leads to a broad peak

• Microvariants may go undetected

Minimise non-template addition

Not overloaded reaction with DNA and final incubation at 72 °C for 45 minutes to add A so everything gets an A on

Allelic dropout

Only one of the two alleles amplifies so there are two possible causes

1. Degradation of the large allele

2. Mutation of the primer site

Allelic dropout example

• 6 is not detected

• At 3’ end, it can’t anneal properly

• It won’t be amplified

• Only the other allele is amplified

Minimise allelic dropout

Make primers to sites that are not polymorphic between individuals and populations

Sizing

• Software looks for predefined pattern of the ladder

• The multi-colour system allows internal size standards to be run in the same gel lane

• Different systems

• DNA fragment peaks are sized based on the sizing curve produced from the points on the internal size standard

• Local Southern method

Promega systems

• Use the ILS 600

• 22 fragments between 60 - 600 bp

Beckman systems

Use 2 different size standards for STR analysis

• CEQ 400 - 22 fragments between 60 - 420 bp

• CEQ 600 - 33 fragments between 60 - 640 bp

Local Southern method

• Requires two standard peaks above unknown and two standard peaks below unknown

• For CEQ 400 size standard, a 165 bp peak would be sized using the 140 bp and 160 bp on one side and the 180 and 200 bp on the other

• It’s used often but not always the best for every situation