Degradation, Contamination and Mixed Samples

Forensic Samples

DNA Sample is Often:• Degraded

– By exposure to conditions/environment/time• Contaminated• Mixed Samples

– Especially in cases of sexual assault• Limited quantity of sample

– Difficult or impossible to obtain more

Degradation of DNA

• When DNA is exposed to environment long complex DNA molecules are randomly broken into smaller pieces

• Degrading DNA:– Water– Nucleases– Sunlight– Microorganisms

Benefits to using STRs

• RFLP genotyping requires high molecular weight DNA

• Because allele sizes are large– More than 10,000 bps

• STRs work much better with degraded DNA because:– Allele sizes less than 500 bps– PCR Amplification step

Quality of DNA Sample

• Can see quality of DNA on agarose gel:

Intact:High Molecular

Weight DNA

Degraded:Smear ofdegradedfragments

Product Size Matters

• STRs work better with degraded DNA• Larger PCR products work less well

– DNA degraded into very small pieces• STRs markers that work:

– Smaller products (100 to 250 bps)• STR markers that may not work:

– Larger products (>250 bps)• Don’t see allele drop out – more likely to

see entire marker showing no genotype

Degraded DNA

• Even severely degraded DNA can still be genotyped with very short markers:

100 150 200 250 300 350 400 450

D13S317 D7S820 D5S818 CSF1PO PentaD

miniSTRs

• Inverse relationship between size of locus and successful PCR genotyping of degraded DNA

• Redesign the primers to generate smaller PCR products

• “miniSTRs”• Moved the primers as close as possible to

the repeat region

CODIS Markers

• 13 CODIS markers have been optimized so that they can be multiplexed together

• Four or more loci are genotyped with each dye – to get all 14 into one reaction

• Separate products based on size:– Some primers are close to repeat region– Produce smaller products– Other primers have large flanking sequence– Produce larger products

miniSTRs

100 150 200 250 300 350 400 450

9246

11254

100 150 200 250 300 350 400 450

993

11102

D16S359 miniSTR Primers

Conventional Primers

STR Repeat RegionD16S359

Using miniSTRs

1. Quality check DNA sample1. Extent of degradation2. By running agarose gel

2. Genotype with conventional markers

3. If necessary go back and re-genotype any loci that were unsuccessful with conventional markers with miniSTRs

PCR Inhibitors

• DNA samples taken from crime scenes often have PCR inhibitors as well

• Inhibitors can:– Interfere with cell lysis– Degrade DNA– Inhibit polymerase activity

• Some inhibitors will remain with the DNA through the entire DNA isolation process

Solutions to PCR Inhibition

One or more of the following techniques:• Dilute genomic DNA

– Dilute inhibitors as well• Add more DNA Polymerase

– Binds the inhibiting molecules– Remaining Polymerase can do its job

• Specific Polymerases work better against specific inhibitors

Solutions to PCR Inhibition

• Specific additives improve polymerization conditions:– BSA – Bovine Serum– Betaine – complex organic– Sodium Hydroxide – neutralizes inhibitors

• Aluminum Ammonium Sulfate– Removes soil from DNA sample

• Additional purification step prior to PCR– Filters may separate DNA from inhibitors

Contamination

• Thanks to the sensitivity of PCR –contamination is a problem

• Especially if a small quantity of DNA sample is contaminated with a large amount of DNA contaminate

• PCR will amplify the DNA that is in higher concentration much faster

• Completely overload the true sample

Contamination

• Contamination of DNA means –accidental addition of another DNA source

• Three primary sources:1. Contamination that exists in the environment

that has nothing to do with the crime2. Contamination between two samples during

analysis3. Contamination of PCR reaction with PCR

product from previous reaction

Contamination

1. Contamination that exists in the environment that has nothing to do with the crime

• Control for this with:– Negative controls in PCR– Reference samples If sample is from people who live in area– Staff Elimination databasesIf sample is from investigators analyzing DNA

Contamination

2. Contamination between two samples during analysis

3. Contamination of PCR reaction with PCR product from previous reaction

• Both of these are controlled by:– Appropriate lab procedures– Removing PCR products away from PCR

reaction set-up– Negative controls

PCR Tubes (Consumables)

• Forensic Science Service (FSS)• Discovered that 11 DNA profiles were

consistently coming up in different cases• Even in negative controls• Analysis of tube manufacturer employees

identified 10 of the 11 DNA profiles belonged to employees

• The PCR tubes were contaminated

Avoiding Contamination

• Staff Elimination database– All people who ever come in contact with

sample– Police, Analysts, Tube Manufacturers

• Reference samples• Using robotics for PCR automation• Negative controls in every set of samples• Well maintained laboratory procedures

Important Distinction• DNA is not found at crime scene

– This simply means that there is no DNA evidence

– Doesn’t prove innocence • DNA does not match DNA sample from

crime scene– If a DNA profile is obtained or can be later

obtained from old evidence– Suspect’s DNA profile doesn’t match– May be because of contamination of evidence

Mixed Samples

• Mixture arises when two or more individuals contribute to DNA sample during the crime

Better chance of identifying that the sample is mixed:

• More markers used• More polymorphic loci• Because more chance that both samples

will be heterozygous – see 3 or 4 peaks

Distinguishing Mixed Genotypes

First must ascertain if DNA profile is showing evidence of a mixed sample

• Ask the following questions:• Do any loci show more than two peaks?• Is there a severe peak imbalance in

heterozygotes?• Does the stutter product seem high?

– More than 15 to 20%

Quantitative Information

• Current technologies• Allow quantifying each peak:

– Either by peak height– Or by area under peak

• This then allows analyst to differentiate:– Heterozygote– Mixed Samples– Stutter bands

Single Source vs. Mixed Source100 150 200 250 300

100 150 200 250 300

> 70%< 15%

Heterozygote Region

Mixture Region

Stutter Region

> 70%< 15%

Too high for stutter? Wrong side for stutter?

Too small to be heterozygote?

Single Source:Heterozygote

Mixed Source:Double

Heterozygote

Possible Genotypes for Mixed Sample

• Four Peaks– Both samples are heterozygous

• Three Peaks– One sample is homozygous or alleles overlap

• Two Peaks– Both samples are homozygous or alleles

overlap• One Peak

– Both samples are homozygous with exact same allele

Interpretation of Mixed Genotypes

• Some genotypes may be hidden– Two samples have same sized alleles

• Difficult to call alleles correctly• Especially when considering artifacts also

exist in same genotypes– Null alleles, stutter bands, etc

• Also, consider that one of the samples in the mixture may be in great excess– Higher concentration

Interpretation of Mixed Genotypes

1. Identify the presence of a mixture2. Designate all allele peaks possible3. Identify number of possible contributors4. Estimate relative ratio of samples

• How much of each DNA sample?5. Consider all possible genotype

combinations• What could DNA profiles possibly be?

6. Compare to reference samples

Interpretation of Mixed Genotypes

Identify the presence of a mixture• Are there 3 or more peaks for multiple

markers?Designate all allele peaks possible• Difficult to remove stutter bands• Try to call any possible alleles evidentIdentify number of possible contributors• Are there more than 4 bands?• Majority of forensic samples are 2 people

Interpretation of Mixed Genotypes

Estimate relative ratio of sample• Examine profile as a whole• Difficult to estimate when an allele is

shared (will look 2:1:1 ratio)• May not be able to interpret genotypesConsider all possible genotype combinations• Calculate all possible DNA profilesCompare to reference samples• Try to determine someone’s profile

Example Mixture

Examine and interpret mixed sample genotype on pages 162-163 of book:

1. Mixture is present (3 and 4 peaks, XY)2. Only two samples (no more than 4)3. Call all alleles – A, B, C, D4. Calculate mixture ratio (2:1)5. Higher peaks belong to male6. Each DNA profile is determined (fig 7.5)

True Chromosomal Abnormalities

• Rare events• Will only present 3 peaks at one region –

all other markers will be normal• Chromosomal abnormality needs to be

confirmed:– Karotyping, more genotyping, sequencing

• Once confirmed may be beneficial– Because these events are so rare– Unlikely anyone else will have same genotype

Low Copy Number

• Ability to obtain a DNA profile from an extremely small amount of DNA

• Less than 100 pg• Methods:

– Increase number of PCR cycles– Amplify DNA first – then PCR– Decrease amount of volume of PCR reaction– Adding more PCR product to analysis– Changing analysis conditions

Low Copy Number

Many possible complications arise:• Allele drop out• Allele drop in (inventing a new allele)• Recovering DNA profiles that have nothing

to do with the events of the crime• Increased risk of contamination

– Since such a small sample is being amplified• Stutter products are enhanced – cannot

separate from true alleles

Working with LCN

• Analyst must work with extreme caution not to contaminate sample

• Everything done in duplicate/triplicate• Negative controls are mandatory

– Throw out any experiments with bands in negative controls

• If suspect’s DNA matches – further experimentation should be done

Common Casework Challenges

D3S1358 TH01 D13S317 D16S539 D2S1338

MIXTURES

DEGRADED DNA

D5S818D13S317

D7S820

D16S539 CSF1PO Penta D

From Butler, J.M. (2004) Short tandem repeat analysis for human identity testing. Current Protocols in Human Genetics, John Wiley & Sons, Hoboken, NJ, Unit 14.8, (Supplement 41), pp. 14.8.1-14.8.22

Loss of signal at larger size loci

More than two alleles at multiple loci

Any Questions?

Read Chapter 8