JS 190- Introduction to STRs- Continued

I. Learning Objectives (C6 Butler)

a. Short Tandem Repeats
1. CE artifacts and Fluorescent Dye multiplexing revisited
2. Biology of STRs- Define-
Balance
Stutter Products
Non-template Addition
Microvariants
Null Alleles
Mutation Rates
 CE artifact:Spikes in formamide blank-
4 colors (S. Myers-CA DOJ DNA)

These are not real DNA peaks

CE artifact: Spikes in one color- no stutter, pull down

 Fluorescent Emission Spectra for Dyes
Filters collect light in narrow range
Overlap is automatically calculated and subtracted using fluorescence “matrix” standards

5-FAM JOE NED ROX

100
Normalized Fluorescent

80

60
Intensity

40

20

0
520 540 560 580 600 620 640
WAVELENGTH (nm)
Laser excitation
(488, 514.5 nm) ABI 310 Filter Set F with color
contributions between dyes
 Dye overlap shown in raw data
Automatically subtracted in processed data (BGYR)
 Short Tandem Repeats:
a subgroup of tandem repeats
(Kuhl and Caskey 1993. Curr. Opin. in Genet. Dev. 3:404)

• Head to tail arrangements of sequence units (4bp),

• Common in genomes (thousands distributed)
• Polymorphic: vary in length by no. of and/or by content of
repeats.
• Stably inherited on a human time scale (for most)
• Well studied b/c others are implicated in Human Diseases and
therefore the subject of clinical studies.
 Trinucleotide Repeats
Implicated in Human Diseases
Sutherland and Richards. 1994. Dynamic Mutations American Scientist 82:157
Disease Chrom Parental REPEAT # Normal # Pre- # full
SexBias of SEQ mutation mutation
severe
form >66%GC
Fragile X X maternal CCG 6-50 50-230 230-2000
syndrome
spino- X ? AGC 11-31 40-62
bulbar
muscular
atrophy
(Kennedy
disease)
myotonic 19 maternal AGC 5-35 50-80 80-2000
dystrophy
Huntingto 4 paternal AGC 9-37 30-38 37-121
n
Disease
spinocere- 6 paternal AGC 25-36 43-81
bellar ?
ataxia type
1
FRAXE X ? CCG 6-25 25-200 200++
dentatorub 12 paternal AGC 7-23 49-754
ral and (mainly)
palliduluys
ian atrophy
 Trinucleotide repeat expansion for
Fragile X syndrome in the FMR-1
gene
 
Copies of CGG 'Phenotype'
 
CGG 6-54 Normal
CGGCGG 50-200 Normal Transmitting Male
CGGCGG 50-200 Daughter
CGGCGGCGG 200-3000 Affected Individual
Advantages of STRs in Forensics
• All of the above and more! Common, polymorphic, stably
inherited, well studied- discrete sizes
• Small size and size range- Useful on highly degraded samples
• Small size range- Less prone to preferential amplification of the
smaller allele
• Multiple STRs provide powerful discrimination
• Abundance permits choice of STRs with non overlapping size
ranges.
• Even for those with overlapping sizes, use of different color
fluorescently tagged primers permit rapid automated analysis.
 Small size and small size range
permit typing of highly degraded
samples
• 73 pathological samples exposed to high
temperature, incineration, explosion and chemical
insult.

Waco disaster: All four loci success 63%, at least 1

locus 83%
• VWFA31, THO1, F13A01, FES/FPS
• Whitaker et al. Biotechniques 19:670
 Multiplexing provides powerful
discrimination
• # Loci Most Common Reference
• 3 1/500 individuals Edwards
Edwards et al. 1994. AJHG 55:175

• 6 1/200,000 AJHG 49:746

• 9 1/300,000,000 (nineplex)Walsh

• 13 (CODIS loci)
• 1/100,000,000,000,000 Walsh 98 JFS
Biological Issues and “Artifacts”
of STR Markers

• Balance of results
• Non-template nucleotide addition- aka. N+1,
aka. 'split peaks', aka. incomplete extension
• Stutter Products- aka. Repeat slippage
• Microvariants – aka. Deletions
• Null alleles- primer binding site mutations
• Mutations
 Balance of results among loci
• In multiplex PCR reactions, some loci may amplify
more efficiently than others. Ideally, individual loci
in a multiplex should not differ in signal intensity
by more than about 10-20%, thereby insuring that
mixtures can, in most circumstances, be easily sorted
out.
• A multiplex which may exhibit perfect signal balance
with pristine DNA may, however, show preferential
amplification with "forensic type" samples,
presumably due to the alteration of the reaction
environment by the addition of contaminants which
co-purify with the DNA.
Balance within and among loci
 Non template directed nucleotide
addition to blunt ends (aka. N+1, split peaks,
incomplete extension)
• Taq polymerase will often add an extra nucleotide to the end of a PCR
product; most often an “A”
• Dependent on 5’-end of the reverse primer
• Can be enhanced with extension soak at the end of the PCR cycle (e.g., 15-45
min @ 60 or 72 oC)
• Can be reduced with new polymerase
• Best if there is NOT a mixture of “+/- A” peaks

• (Clark,J. NAR 16:9677, Hu. 1993. DNA and Cell Biol. 12:763.)

A
A
 Non template directed nucleotide
addition to blunt ends
• A property of the Taq (and other DNA polymerases), not
specific to STRs where an extra nucleotide is added to the
3'OH end of blunt ended double stranded DNA
Problem when it is not 100% because peaks (bands) are
split (two peaks for the same product, one base pair
apart). It is sequence specific, so not all loci will exhibit,
and is effected by rxn conditions (eg Mg2+).
• For STRs resolved by adding an extension at the end of
thermal cycling. The extension to favor nt+ is currently
done at 60C for 30 minutes. The lower temp is used to
reduce 'breathing' between the template and extending
strand. The choice of primer sequence can influence the
amount of nt+.
 Higher Levels of DNA Lead to
Incomplete Adenylation
DNA Size (bp)

-A
Relative Fluorescence (RFUs)

off-scale
+A
10 ng template
(overloaded)

D3S1358 VWA
FGA

2 ng template
(suggested level)
Impact of the 5’ nucleotide
on Non-Template Addition
+A +A 5’-ACAAG…

Last Base for Primer

Opposite Dye Label

+A +A
-A -A 5’-CCAAG…
 Stutter or Repeat Slippage
• Definition: Peaks that show up primarily one repeat
less than the true allele as a result of strand slippage
during DNA synthesis (-n where n=1 repeat = 4bp).
• Faint peaks or bands which are sized as true allele
-n, -2n, -3n…). Each successive stutter product is less
intense (allele > repeat-n > repeat-2n>repeat-3n)
• All DNA polymerases seem to do it (in fact this
phenomena occurs in genetic diseases resulting
from repeat expansion).
• In most forensic STR systems we usually only see
the repeat-n stutter product
 Stutter as it correlates to allele
size (eg number of repeats)
• Levels of repeat slippage vary for different loci and
even for the different alleles of a particular locus.
• Amount of repeat slippage appears to be greater in
larger alleles with more repeats and less in those
that are smaller. Longer repeat regions generate more
stutter. That is, a 20 repeat allele will generally have
more stutter than a 10 repeat allele
• Amount of slippage for a given sized allele
appeared to be quite reproducible.
 Stutter as it correlates to unit
size
(eg the number of bases in a single repeat)
• Stutter is not as bad with larger repeat unit sizes.
• Very bad with small size- di-nucleotide
repeats.
• Not as bad with larger size - tetra and penta
nucleotide repeats
• (dinucleotides > tri- > tetra- > penta-)
 STR Alleles with Stutter
Products
DNA Size (bp)

D8S1179
Relative Fluorescence Units

D21S11 D18S51

Allele

Stutter
Product
6.3% 6.2% 5.4%
 Microvariant Alleles
• Not all alleles have full length repeat units
• Alleles with partial repeat units are
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
 Microvariants
• Defined as alleles that are not exact multiples of
the basic repeat motif or sequence variants of the
repeat motif or both
• May exist as insertion, deletion, or base change
• Sequence variation can occur within repeat, in the
flanking region, or in a primer binding site
 Detection of a Microvariant
Allele at the STR locus FGA

1 = S25-L25 = 244.34 - 244.46 = -0.12 bp

2 = SOL - L28 = 257.51-256.64 = +0.87 bp

28.1
28.1
c = |1 -2| = |-0.12-0.87| = 0.99 bp
 Three-Peak Pattern at D18S51
AMEL
D8S1179 D21S11
D18S51
 Null Alleles
• 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 different results on
samples originating from the same source
• This phenomenon impacts DNA databases
• Large concordance studies are typically performed
prior to use of new STR kits
Impact of DNA Sequence Variation in
the PCR Primer Binding Site
Heterozygous alleles
are well balanced

6 8

Imbalance in allele
peak heights

6
8

*
8

Allele 6 amplicon
has “dropped out”
 Mutation Observed in Family Trio
Mutations may be detected in children
Occur at approx 0.1-0.3% at each STR locus and appear to show a
paternal bias- Dads STR change more frequently than Moms

14,18 15,17 14,18 15,17

15,18 13,17

Normal Transmission of Alleles Paternal Mutation

(No Mutation)
 Measured Mutation Rates
STR Locus Maternal Meioses (%) Paternal Meioses (%) Null Alleles (%) Multi-Banded (%)

CSF1PO 14/47843 (0.03) 311/243124 (0.13) 2/42020 (<0.01) None reported

FGA 7/8253 (0.01) 555/189973 (0.29) 2/1104 (0.18) None reported

TH01 5/42100 (0.01) 12/74426 (0.02) 2/7983 (0.03) 0/2646 (<0.040)

TPOX 2/28766 (0.01) 10/45374 (0.02) 11/43704 (0.03) 13/42020 (0.03)

VWA 20/58839 (0.03) 851/250131 (0.34) 7/42220 (0.02) 1/6581 (0.02)

D3S1358 0/4889 (<0.02) 9/8029(0.11) None reported None reported

D5S818 22/60907 (0.04) 194/130833 (0.15) 3/74922 (<0.01) None reported

D7S820 14/50827 (0.03) 193/131880 (0.15) 1/42020 (<0.01) 1/406 (0.25)

D8S1179 5/6672 (0.07) 29/10952 (0.26) None reported None reported

D13S317 33/59500 (0.06) 106/69598 (0.15) 52/62344 (0.08) None reported

D16S539 12/42648 (0.03) 40/48760 (0.08) 3/52959 (<0.01) 0/1165 (<0.09)

D18S51 8/8827 (0.09) 29/9567 (0.30) None reported None reported

D21S11 12/6754 (0.18) 17/6980 (0.24) 1/203 (0.49) None reported

http://www.cstl.nist.gov/biotech/strbase/mutation.htm
*Data used with permission from American Association of Blood Banks (AABB) 1999 Annual Report.
 Review of STRs
Intro to STRs
– Head to tail arrangements 4 bp repeat units
– Polymorphic, Common, Stably Inherited, Implicated in
Diseases
– Advantages- Discrete, Small- less prone to PA, Useful on
highly degraded DNA, Ability to Multiplex , Provide powerful
discrimination.
– STR biological artifacts- stutter, adenylation, microvariants,
null alleles, mutations
– Results are interpreted by reproducibility, size of the resulting
fragment, spectral properties, stutter, and size of peak
(balance within and among loci).
– Multiplexing STR loci provide powerful discrimination