STR Biology

8/7/2019 STR Biology

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Biology of STRs


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Artifacts in Genotyping STRs

A number of artifacts are possible:

Stuttering Non-template additions

Microvariants Three peaks

Allele dropouts

Mutations

All interfere with reading a DNA profile

accurately and consistently


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Stuttering

Stuttering is caused by the very structure

of the STRs that make them good markers They are repeats

That are highly polymorphic Stutter product is a band that has the

wrong number of repeats Either one repeat more or one less

Caused by strand slippage


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Strand-slippageATGCGGCGGCGTGTGTGTGTGGCG

TACGCCGCCGCACACACACACCGCCG53

ATGCGGCG

TACGCCGCCGCACACACACACCGCCG

53

CGTGT

GGT

GT

GT

DNA Replication

Or PCR

Elongation

ATGCGGCGGCGTGTGTGTGTGGCGGC


53

GT

ATGCGGCGGCGTGTGTGT


53

GT Misalignment


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Strand Slippage

Occurs during extension step of PCR

The newly formed strand of DNA skipsone repeat unit starts complementary

base pairing with next repeat Pushing out a non-base paired loop from

the template strand of DNA

Usually causes a deletion of one repeatunit therefore band will be one unit

smaller than true genotype


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Strand Slippage

Naturally this is the mechanism that

makes repeats polymorphic When it happens during PCR it can

produce a band that is not real: Genotype will be wrong

One repeat unit lower or higher than reality

Rarer in Tetranucleotides than any otherrepeats which is why tetras are used


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Amount of Stutter Product

Stutter is usually rare

Therefore might show a small bump - canusually be differentiated from a true band

Earlier in PCR reaction strand slips More stutter product will be produced

Or if genotyping protocol doesnt work welltrue band may be very low

Difficult to separate stutter band from true

band


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Stutter Products

Stutter Stutter Stutter ?

Call these genotypes:


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Calling Alleles

Biggest problem with stutter bands:

They are the same size as a real allele!

Especially difficult if you know the DNA

sample is mixed Or you are unsure whether sample has

been contaminated

Difficult to determine:

Stutter band

Minor allele (because less DNA)


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13 CODIS STR Loci

All produce some stutter products

Longer alleles produce more stuttering Why does this make sense?

Stutter percentages for Tetranucleotides: From Less than 1 %

Up to 15% - of the true allele size

Therefore always calculate percentage ofsmall bands peak height

Be sure < 15% height of large band


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Reducing Stuttering Products

Changing PCR conditions

Faster DNA Polymerase Faster it works, less chance for slippage

STRs with longer repeats (> 4 bps) More difficult to skip past repeat

STRs with imperfect repeat units Complex and compound repeats

More difficult to skip past repeat if next repeat

unit sequence is different


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Summary of Stutter Products

One repeat unit more or less than real

allele peaks Less then 15% real allele height

Quantity of stutter band depends on: When in PCR reaction first slippage occurs

Allele size (bigger alleles, more stutter)

PCR Conditions

Polymerase used

Repeat length and sequence


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Non-Template Additions

Polymerase often adds an extra

Adenosine to the end of the newly formedsequence

Not a part of the template sequence Makes PCR product one base longer than

actual sequence

If your PCR reaction forms both +A and -Aproducts then your band will be wide


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Want to have peaks as clear as possible

Therefore want all PCR products to beidentical

Either all +A or all -A Imagine case where you were genotyping

a dinucleotide, with stutter, and half theproducts were +A and half were -A

Impossible to separate genotypes


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Set up PCR conditions so that every

product will be +A Conditions:

Final extension for 10 mins Allows all products to be fully adenylated

Primer ends in a guanosine

Commercially available kits turn everyallele (and ladder) into +A


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Overloading Sample

Signal on gel is too strong will be difficult

to call May result in a split peak

Or a peak that is off scale Caused by:

Too much DNA sample in PCR reaction Primer concentrations too high

Why DNA quantification is so important


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D3S1358 VWA FGA

-A

+A10 ng

template(overloaded)

2 ng template(suggested level)

DNA Size (bp)

RelativeF

luorescence(RFUs)

off-scale

Figure 6.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition 2005 Elsevier Academic Press

Non-Template Additions and

Overloading Samples


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Microvariants

Remember these are variants of the

repeat that are not a full repeat unit Example TH01 9.3 allele

As opposed to stutter allele microvariantsare not same size as expected allele

Problem is determining whether there isa true microvariant in the person

Or you are seeing a normal band being

shifted over for some genotyping reason?


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Microvariants

1. True microvariants must be validated to

happen in many samples Even if variant is rare it must show up in

more than one individual to be considered a

true microvariant

2. Exact distance in base pairs should be

calculated 9.3 means 9 repeats plus 3 bases

Always calculate in bases exactly how offthe microvariant is


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Sequence Microvariants

Sometimes there are also sequence

differences in these polymorphisms aswell as length differences

The only way to genotype a sequencevariant is to sequence the PCR product

Not necessary for Forensics because you

are simply matching genotypes

These variants are not important for

Forensics analysis


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Peaks outside of the Ladder

Sometimes you will see a peak that it

outside of the expected range for anymarker (between markers?)

What could cause this? Unsuccessful PCR product

Primer dimers or etc.

Person really has a new allele

Check with different set of primers

Sequence new allele and region


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Three Peaks

Sometimes three bands may be seen

What could cause three bands? Stuttering

Mixed or contaminated samples Genotyping error

True duplication or extra chromosome in the

individual

Need to validate what is seen in gel


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Three Peaks

1. Check other markers in panel:

1. Is there evidence of mixed or contaminatedsamples in any other markers?

2. Check database information for this

marker:1. More than 50 tri-allelic patterns have been

reported as possible with 13 CODIS loci3. Sequence or genotype this region:

1. Is there truly a duplication or extrachromosome in this person?


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Allele Dropout

Most worrisome problem

May call a person homozygous whenreally they are heterozygous

What can this be caused by?

Larger allele is not amplified successfully

Primer site mutation

Rare with chosen tetranucleotides: Alleles are very similar in size

Primers have been optimized and chosen inregions that are very stable


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Avoiding Allele Dropout

Chose primers carefully

Work with polymorphisms that have allelesof similar size

Always check genotypes with Hardy-Weinberg Equation

Make sure you see the expected number of

heterozygotes population wide

Most commercial kits have taken care of

all these issues


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Fixing Allele Dropouts

Add a degenerate primer

Extra primer with known polymorphism Three primers total will be added

Lower annealing temperature Reduce the stringency of primer binding

Remember that with Forensics whatmatters is matching genotypes

As long as allele always drops out, dont have

a problem


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Mutations

STRs do mutate at an expected mutation

rate over time Mutation may cause:

New Alleles Change primer binding regions

Sequence changes (less important)

Very rare events

Can be validated by examining families


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Mendelization of Alleles

Using family members to determine which

alleles are possible If you know parents alleles then there are

only so many genotypes possible forchildren

Mendels law of segregation

All STRs have been genotyped on CEPHfamilies huge family sets from Utah


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Mendelization of Alleles

3/148/12

3/8 8/14 3/12

2/9 5/11

10/112/11

As always must validate mutation

By sequencing or regenotyping


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Mutation Rates

Mutations rates of 13 CODIS have been

calculated over thousands of meioses All 13 are between 1 to 5 per 1000

generational events

Highest mutation rates:

Markers that are most polymorphic

Lowest mutation rates:

Markers that are least informative


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Impact of Mutations

Paternity testing

Can cause problems Because father may not match true child if

genotype has change in child

Compare many STR loci

Identity matching

Will not cause a problem

Because mutation will be consistent over a

persons lifetime and in all tissues


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Genotyping Errors

All the previous were artifacts that can be

explained However the problems you really worry

about are unexplained errors

Especially if sample may be:

Contaminated

Mixed samples

Need to always validate any artifact

Be sure its not genotyping error


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Any Questions?

Review Chapters 1 6

Email me at least 2 questions you

have about the first 6 chapters

Next class will be review for Exam

Exam One February 5th

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STR Biology