Forensic Biology: Lecture 5 – STRs & SNPs in Forensic Analysis

Homozygote

both alleles are the same length

Heterozygote

alleles differ and can be resolved from one another

Homozygote

both alleles are the same length

Heterozygote

alleles differ and can be resolved from one another

STRs are the preferred genetic markers because they:

are highly variable within various populations.

Simple repeats

Variation is due to differences in the number of repeats

Simple repeats with non-consensus alleles

In addition to number of repeats, some of the repeats may be incomplete

Microvariants

STRs with altered repeat units

Compound repeats

Two or more types of repeat units

Compound repeats with non-consensus alleles

Two or more types of repeat units and some of the repeats may be incomplete

Complex repeats

They can have several types of repeat units, with interruptions between them

replication slippage.

During replication the newly synthesized DNA strand can be displaced, and a loop of unpaired DNA is formed between the old and new DNA strands. The result is that after replication, there may be a gain or loss of a repeat unit

STRs show two important differences with respect to other genetic markers (SNPs)

1. A much faster mutation rate (the average is around 10-3).
2. Instead of only two alleles, they typically have multiple allelic forms.

SNPs are

single base pair changes in DNA

How often, on average, do SNPs occur?

once every 300 base pairs

Polymorphism

The coexistence of two or more distinct forms in the same population.

There were originally 13 STRs for identification, but the CODIS panel newly upgraded to:

20

Genotyping of the CODIS STRs (plus Amelogenin for sex determination) is done by:

capillary electrophoresis

RFLPs are

varying DNA sequences that are recognized by restriction enzymes

Endonucleases

enzymes that cut RNA or DNA at specific sites; restriction enzymes

Exonuclease

enzyme that removes successive nucleotides from the end of a polynucleotide molecule

Genotyping the CODIS panel requires just 1 or 2 days, while the RFLP markers required:

1 week or more

Probability of a random match using 13 CODIS STR markers:

1 in 594 trillion

Multiplex PCR is:

the simultaneous amplification of several markers in the same PCR reaction

Capillary Electrophoresis (CE)

A method of separating DNA samples based on the rate of movement of each component through a gel-filled capillary while under the influence of an electric field

Internal size standards

combined with PCR product; set of DNA fragments of known size used to correlate results from run-to-run, allow allele sizing.

Allelic ladder

set of fragments representing all possible alleles of a repeat locus (STR)

STR genotyping is performed by comparison of sample data to:

allelic ladders

Ability to identify sample as male or female is useful for:

- Sexual assault cases
- Missing persons cases
- Mass disaster investigations

Amelogenin assay is the most useful for sex determination because:

it can be performed in conjunction with STR analysis

Rare mutation where the Y chromosome amplicon is absent, m ost common in South Asian populations:

Male samples falsely appear to be female

Mutation in primer binding sites on X chromosome causes amelogenin X allele dropout:

Only Y amplicon is present

Most abundant markers in the human genome:

SNPs

dbSNP

database for human SNPs maintained by the NCBI

Genotyping SNPs requires

use of PCR to amplify the region where the SNP is located

SNPs classified into four general uses:

- Human identification
- Ancestry informative markers (AIMs)
- Lineage informative SNPs
- Phenotype informative SNPs

SNPs have a low mutation rate and are therefore more likely:

to become fixed in a population making them population specific, which is helpful in determining ancestry of perpetrator or found remains.

Disadvantages of SNPs

They are diallelic, meaning they have a lower power of discrimination than STRs, which are multiallelic.

Advantages of SNPs

Work better for severely degraded DNA samples; amplicons can be as small as 40-50 bp, whereas STRs amplicons are typically between 100-500 bp.

What is RFLP mostly used for?

Genome mapping and in variation analysis (genotyping, paternity tests, hereditary disease diagnostics, etc.)

The McSNP Principle

In addition to distinctive lengths, DNA fragments have diagnostic melting points where the two strands separate.

TaqMan and molecular beacons:

methods based on the hybridization of an oligonucleotide (also known as a probe) to the sequence where the SNP is located (target sequence).

TaqMan Probe

Two probes per reaction; each is complementary to a different allele; labeled with a dye (fluorescent molecule) and a quencher (absorbs the energy of the dye); a perfect match of the probe to the target DNA will give a signal.

Molecular Beacons

Two probes are used, each complementary to a different allele; in the absence of the target DNA, the probe has a stem-loop structure; in the presence of the target DNA, the probe hybridizes to the complementary sequence, the quencher and reporter are driven apart, and there is fluorescence.

mini-sequencing

primers flank the polymorphic site of interest; two nucleotides labeled with different fluorophores are added to the primers by the action of Taq polymerase; the genotype is detected by the difference in fluorescence.