Forensic Issues in DNA profiling

How does environmental exposure affect DNA molecules?

Randomly breaks it into smaller pieces through hydrolsyis

What are the "enemies" for the survival of intact DNA?

Water, microorganisms, and nucleases

What is the affect of nucleases on DNA?

Nucleases cleave the phosphodiester bonds between DNA, breaking it into smaller pieces

How can agarose gels be used to evaluate the quality of a sample?

Degraded dna will result in smearing

What is a sign of good quality DNA on an agarose gel?

High relative molecular mass and a tight band

What was the issue with prior DNA typing techniques, such as RFLP and VNTR?

They required large alleles with thousands of bps, which wouldn't be possible with degraded dna

What does RFLP stand for?

Restriction Fragment Length Polymorphism

What does VNTR stand for?

Variable Number Tandem Repeats

How do STRs address some of the previous issues in typing degraded DNA?

Since STRs require less bps than prior techniques, they work better on degraded DNA

What must be present in order for PCR amplification to still occur?

Primer sites and full sequence in between

What is the "ski slope effect" ?

Occurs when dna is degraded and larger amplicons are lost or reduced, resulting in smaller signal peaks

What are miniSTRs? How do they address the issue of degraded DNA?

Cuts out the flanking region and anneals the primers directly to the repeatable sequence. Makes STRs even smaller.

What are some of the issues with MiniSTRs?

You would need a lot more dye channels, and there's some issues with redeveloping primers.

What do inhibitors do to DNA samples?

Interfere with cell lyses, causes dna degradation or capture, and inhibit polymerase activity

What are some examples of inhibitors in outdoor scenes?

Soil, sand, wood, leaves

What are some examples of imhibitors in interior scenes?

Textiles, dyes, leather, and wood

What is an example of an inhibitor in blood?

heme

What are inhibitors in tissue and hair?

melanin

What are inhbitors in feces?

bile salts

What is the inhbitor in soil?

humic compounds

What is the inhibitor in urine?

urea

What is an inhibitor in blue jeans?

textile dyes

Why can inhibition sometimes be mistaken for degradation?

Inhibition also results in a ski slope effect due to the decrease in effeciency

What is the BEST way to address inhibitors?

Purifying before amplification

What is the difference between dna degradation and dna inhibition?

DNA degradation refers to the breakdown of dna molecules into fragments while dna inhibition is the process of preventing dna replication from occuring

What are some alternative solutions to inhibitors?

Dilution, more polymerase, additives such as BSA (bovine serum albumin)

How does BSA help decrease inhibition?

Binds to the inihibitors and prevents them from interfering with DNA polymerase

What quantity of dna do you need for profiling?

0.5-1 ng

When is a sample considered "low template" ?

Samples with less than 100 pg

What are some strategies of improving testing sensitivity in low template DNA?

Increase number of pcr cycles or increase CE injection, post pcr purification, reduced volume pcr, nested pcr, mtdna testing

What occurs in post pcr purification?

Removes salts and other impurities so there's more "targets" in injection

What is reduced volume PCR?

less than 25 microliter reactions. Allows for highly concentrated sample

What is nested pcr?

PCr variation that uses two sets of primers in successive reactions

Why is mtDNA considered an alternative in cases of low template dna?

There's a higher number of mtDNA molecules per cell, improving the chances of obtaining a result

What is one of the major challenges when attempting to analyze ltDNA?

Stochastic (random) sampling effects occur in early pcr cycles due to the difficulty of primers finding a binding site

What is allele drop in?

Additional alleles from sporadic contamination

What is allele/locus dropout?

Allele present in the original sample fails to amplify due to stochastic effects

What is heterozygous peak imbalance?

One alelle may be preferentially amplified resulting in a peak imbalance exceeding 60%

What is replicate analysis?

Helps address alelel drop in/out by performing replicate pcr amplifications. Alelles that occur in more once are deemed "reliable" and a consensus profile is created