JS 115- Introduction to STRs- Continued
I.
Pre class activities
a.
Review Assignments and Schedules
1.
Assignment- Read Chapters 6 and 7 Butler, Ch 7 Rudin
2.
Optional assignment- Read Scientific American article on
microsattellites- See Lee for copy- 500 word summary with
3 Q ad 3 A
II. Learning Objectives (C6 Butler)
a.
Short Tandem Repeats
1. CE artifacts and Fluorescent Dye multiplexing
revisited
2. Biology of STRs- DefineBalance
Stutter Products
Non-template Addition
Microvariants
Null Alleles
Mutation Rates
CE artifact:Spikes in formamide blank4 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
80
60
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
Fragile X
syndrome
spinobulbar
muscular
atrophy
(Kennedy
disease)
myotonic
dystrophy
Huntingto
n
Disease
spinocerebellar
ataxia type
1
FRAXE
dentatorub
ral and
palliduluys
ian atrophy
Chrom
X
Parental
SexBias of
severe
form
maternal
X
REPEAT
SEQ
# Normal
# Premutation
# full
mutation
>66%GC
CCG
6-50
50-230
230-2000
?
AGC
11-31
19
maternal
AGC
5-35
50-80
80-2000
4
paternal
AGC
9-37
30-38
37-121
6
paternal
?
AGC
25-36
X
12
(mainly)
?
paternal
CCG
AGC
6-25
7-23
40-62
43-81
25-200
200++
49-754
Trinucleotide repeat expansion for
Fragile X syndrome in the FMR-1
gene
Copies of CGG'Phenotype'
CGG
CGGCGG
CGGCGG
CGGCGGCGG
6-54
50-200
50-200
200-3000
Normal
Normal Transmitting Male
Daughter
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
• 3
Most Common
1/500 individuals
Reference
Edwards
Edwards et al. 1994. AJHG 55:175
• 6
• 9
1/200,000
1/300,000,000
AJHG 49:746
(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
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
A
A
•
(Clark,J. NAR 16:9677, Hu. 1993. DNA and Cell Biol. 12:763.)
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
Relative Fluorescence (RFUs)
DNA Size (bp)
-A
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 > repeat2n>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
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
Relative Fluorescence Units
DNA Size (bp)
D8S1179
D18S51
D21S11
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
c = |1 -2| = |-0.12-0.87| = 0.99 bp
28.1
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
15,18
Normal Transmission of Alleles
(No Mutation)
14,18
15,17
13,17
Paternal 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