DNA Forensics Exam 3

familial search

\-primarily state by state (not national)

\-search the convicted offender index to see if relatives of the suspect

low-stringency search

of the state offender database to look for matches that might indicate that a crime

\-neither parent-offspring pairs nor sibling pairs are expected to share genotypes

\-must look at shared alleles

\-(CODIS looks at genotypes, this looks at alleles)

\-Hits are LEADS not EVIDENCE

Allele sharing for a 10-locus STR multiplex kit

• To be considered siblings the dna must share some alleles more than unrelated random people

• for parent-offspring they must share at least one allele at every locus

SNP

single nucleotide polymorphism

\-means not all the people in the population have the same nucleotide at a specific position in the genome

\-2 alleles per marker

\-much lower power to discriminate than STRs(need >50 SNPs to achieve discimination equivalent to 15 STRs)

Disadvantages of SNP

huge current database of STR profiles

SNP versatility

\-more than just identity testing

\-huge number of human SNPs already mapped

mixtures

by far the hardest problem in interpretation of STR profiles

\-Noise, stochastic effects, peak-height imbalance, stutter

Simple analysis IF...

\-DNA is from only two sources - no more than 4 peaks at any locus

\-two sources are unrelated and have no or few shared alleles

Steps of mixture analysis

6 steps

1. Identify the presence of a mixture

2. Designate allele peaks

3. Determine number of contributors (no more than 4 alleles at each locus means 2 contributors)

4. Estimate relative ratio of contributors

5. Consider all possible genotype combinations

6. Compare reference samples

Mixture and reference samples (victim)

From victim:

* Use reference sample to help analysis

\

Mixture and reference samples (unknown contributors)

Unknown contributors:

* Analyze mixture sample before looking at references

SNP locus

one nucleotide beyond boundary

If amelogenin X\=y

probably two male contributors

Familial searches

\-does not produce evidence, only produces LEADs

\-uses the state convicted offender index

Amendment IV

The right of the people to be secure in their persons, houses, papers, and effects, against unreasonable searches and seizures, shall not be violated, and no Warrants shall issue, but upon probable cause, supported by Oath or affirmation, and particularly describing the place to be searched, and the persons or things to be seized.

Familial searching summary (low stringency search)

Looking to match as many alleles as possible, not focused on genotypes

Familial searching summary (hundreds of hits)

• hundreds of hits filtered by sex, age, geography

• Remaining hits treated as normal investigative leads

Familial searching summary (Laws)

\-No uniform laws or rules in the U.S. \n -Common in some places (Colorado) \n -Tightly regulated in other places (California)

\-Probably done without any legislative or judicial oversight in other places

Familial searching summary (Privacy and Bias)

Many innocent people will be treated as suspects just because they share STR alleles with a criminal (related or not)

5\.Bias concerns

SNP mapping

Lotta effort devoted to this because SNPs can be found that are tightly linked to disease phenotypes

Next gene sequencing

5' end of bead that has 50 identical bp in everyone is attached to bead

\~600,000 copies of the probe are attached to the bead

One bead has only 1 probe type (sequence)

\-\*only one base will be added to the 3' end of the oligonucleotide probe

Only use ddntps (terminators

Next gene sequencing cont.

• Color determines which allele and homozygous or heterozygous(mixed color = hetero)

• The camera records the color of every bead.

• The computer knows the identity of each bead (which SNP locus is attached)

• By reading this one plate full of beads, we can determine a person's genotype at ~700,000 SNP loci all at once

Facts to know about SNP genotyping

• Each microbead has many identical oligos attached

• Single-stranded genomic DNA is annealed to the probe oligos

• DNA polymerase is used to extend the probe oligo, using the genomic DNA as the template

• Every Nucleotide Triphosphate is a terminator

• Only one new base ever added

• The fluorescent color attached to the single base added tells the allele at that SNP locus

DNA info*

DNA is inherited in blocks defined by recombination events. We can see this by looking at a sufficiently dense sample of SNPs.

Relationship testing

• paternity

• missing persons

• disaster victim identification

• historical analyses

obligate paternal allele

allele that could not have come from the mother

Paternity test

A test to identify an individual's father by DNA analysis.

* not done on just one locus, but one locus that doesn't have the obligate paternal allele is enough to exclude someone from being the father

Typical paternity test

*Inclusion (or 'not excluded')

• -If all of the obligate paternal alleles in the child are present in the Alleged Father

  *Exclusion

• If some or all of the obligate paternal alleles in the child DO NOT have corresponding alleles in the Alleged Father

Paternity index

PI= x/y

• x=probability that Alleged Father transmitted the obligate paternal allele to the child

• if homo x=1 and hetero x=0.5

• Y = the frequency of the obligate paternal allele in the general population

• both x & y are conditional probabilities

• Result state that (answer) is more likely to have recieved obligate paternal allele from alleged father than random man

str familial search

• low stringency search of codis covicted offender index

• getting alleles and look back at first relatives

• look for shared alleles

• then circumstantial evidence

SNP familial search

using 23 and me or ancestry and looking at snp loci to look at close or more distant relatives\`

Next gen sequencing cont.

• refers to a collection of different technologies

• all allow for massively parallel sequencing

• 1000s to 1,000,000s simultaneous reacations

• ex: ilumina

Ilumina needs

• DNA polymerase

• terminators

• Fluorescent labels

• microfluidic technology

• computer imaging technology

• one primer

Ilumina sequencing

• only use terminators, but the termination is reversible. Attach another molecule to the 3' oh that can be cleaved

• day 23

Legal standard for proof of paternity

• completely arbitrary

• US: often CPI> 100

• Europe: CPI > 1000

Who does paternity testing

• NOT government agencies

• *done by private industries

• AABB- American Association of blood banks is the regulating body for the DNA paternity and family relationship testing industry

What are we looking for in mixture electropherograms

GENOTYPES, not alleles

Mixtures - 2 people

2-person mixtures are easiest to interpret when there is a large difference in the amount of DNA contributed by the two sources

DVI (Disaster Victim Identification) 3 challenges

1. Technical (Obtaining sufficient DNA)

2. Mathematical/statistical (Assessment of confidence or weight of evidence)

3. Organizational (Having a plan and being prepared)

DNA extraction from bone

• grind bone fragment into fine powder

• remove calcium ( demineralization)

• can be done with acid, but that destroys DNA

• can be one with chelating agent

DNA exctraction from bone new

The "total demineralization technique" uses a much higher concentration of EDTA compared to earlier protocols

• new .5 M EDTA(chelating agent)

• sequesters large amount of ca++

DNA extraction after demineralization

• demineralized powder treated with protease

• followed by organic or solid-state extraction

DNA extraction from bone old

• Old = 50 mM EDTA

• Typical for any DNA extraction buffer

• Purpose is to sequester Mg++ ions, to prevent the action of any DNase enzyme

Damaged dna with PCR

If you want to improve the chance of getting useful information from damaged DNA, make the PCR targets as small as possible

Mini-STRs

• used specifically for LCN PCR and degraded samples

• PCR products (=amplicons) designed to be as small as possible

• smaller targets are more likely to remain intact in damaged/ degraded samples

• -**some are new primers for existing CODIS STR loci

limitations of mini-strs

• Analysis limited to 5 loci per reaction

• Limited multiplex capability

• Some mini-STRs are "new" loci

• Require new validation studies

• Cannot upload profile to CODIS

• Limited data on allele frequencies in the population

Recovery of Genetic Information from Degraded Disaster-Victim Samples

1. More information than from regular STRs, from damaged samples But less information than regular STRs from undamaged samples because fewer loci will be examined

2. mtDNA : More copies per cell, so better chance for there to be some undamaged molecules. Much less informative than STRs, only informative about maternal lineage

State the fundamental question asked in paternity testing with respect to court cases.

How much more likely is it that the child obtained the Obligate \n Paternal Alleles from the Alleged Father, rather than from a \n Random Man?