Biology of STRs

Biological artifacts of STR markers

• stutter products

• non-template nucleotide addition

• microvariants

• null alleles

• mutations

Stutter products contain ———than the main allele

one less repeat unit (4bp less than main allele)

larger alleles have——stutter

larger

similar sized alleles may have——-stutter

different

reduced stutter when

homogenous run of repeats interrupted

stutter products form when

taq polymerase is replaced (after 50-60 bases) slipped strand mispairing

dinucleotide repeats have

a LOT of stutter

stutter products

peaks that show up primarily one repeat less than the true allele as a result of strand slippage during DNA synthesis. (typically less than 15% of main allele)

stutter between loci

less pronounced with longer core repeat unit sizes

stutter within a locus

larger alleles with more repeat units generate more stutter

each successive stutter product is ——-intense

less

stutter is reduced when

repeat unit homogeneity is interrupted

stutter peaks make mixture analysis——difficult

more

taq polymerase will often

add an extra nucleotide to the end of a PCR product (most often A)

Added nucleotide to the end of a PCR product can be enhanced with

extension soak at the end of the PCR cycle (15-45 min @ 60 or 72 C)

(best if there is not a mixture of +- A peaks)

Added nucleotide to the end of a PCR product can be reduced with

new polymerase

(best if there is not a mixture of +- A peaks)

Higher levels of DNA lead to

incomplete adenylation

Microvariant alleles

contain incomplete repeat units

• may occur because of insertion/deletion

non-consensus alleles

fall between alleles with full repeat units

how are microvariant alleles designated

by the number of full repeats and then a decimal point followed by the number of bases in the partial repeat

– Example: TH01 9.3 allele

• (AATG)6(-ATG)(AATG)3

Null allele

Allele is present in the DNA sample but fails to

be amplified due to a nucleotide change in a

primer binding site

allele dropout is a problem because

a heterozygous sample appears falsely as a homozygote

two PCR primer sets can yield——results on samples originating from the same locus in null alleles

different

null alleles impact

DNA databases

null alleles are typically identified through

concordance studies

– Large population studies

– Comparison of different kits

Solutions to null alleles

Add additional PCR primer to the assay

– Can hybridize properly to alternate allele

when it exists in a sample

– Called ‘degenerate primers’

Or primers re-designed to avoid

mutation

– Not always possible with commercial kits

most common method of sex chromosome id

amelogenin

• gene that codes for proteins found in tooth enamel

• primers flank a 6bp deletion w/in intron 1 on the X homologue

• 105 and 112 bp products

  • X and Y chrom respectively

Amelogenin dropout

Y dropout

• rare deletion of amelogenin gene

  • cause Y amel product to be absent

  • XY appears as XX

  • More common in Indian populations

    X dropout observed in XY samples

  • Rare polymorphism in primer binding site