Download 51312307

Multistep microsatellite mutation in the maternally
transmitted locus D13S317:
a case of maternal allele mismatch in the child
Devinder Singh Negi . Mahfooz Alam .
S. Annapurna Bhavani . Javaregowda Nagaraju
Present by…
Nonglak Sinkhan
ID 51312307
Introduction
Microsatellite หรื อ
Short Tandem Repeats (STR)
Y-Chromosomal DNA
X-Chromosomal DNA
Mitochondrial DNA
Microsatellite mutation
Purpose
Materials
Sample
Blood samples at the Laboratory of DNA
Fingerprinting Services of Centre for DNA
Fingerprinting and Diagnostics, Hyderabad,
India
Method
Extraction
The quantity of DNA
DNA samples
Phenol/choroform
DNA samples
Amplification
 STR
AmpFlSTR Profiler Plus,
Identifiler (Applied Biosystems, Foster City, USA)
PowerPlex (Promega Corporation, Wisconsin, USA)
 X chromosome
published primer sequences
 Mitochondrial DNA hypervariable regions HV1 andHV2
published primer sequences
Amplification
50-ng DNA
200-µM dNTPs
6 pmol primer
2.5-U AmpliTaq Gold
1xPCR buffer
1.5-mMMgCl2
PCR
Thermal cycling condition :
ABI GeneAmp PCR system 9700
(Perkin Elmer,Norwalk CT)
Denaturation
90ºC ถึง 95ºC
Annealing
50ºC ถึง 55 ºC
Extension
70 °C ถึง 75 ºC
GeneScan and genotyping
ABI PRISM® 3100 Automated Genetic Analyzer
(Applied Biosystems, Foster City, USA)
Sequencing of D13S317 alleles
DNA samples of the suspiciousfather, mother and
questioned child
PCR allelic product of D13S317 locus
purified
6% polyacrylamide gel electrophoresis (PAGE)
Eluted from the gel
Elution buffer (0.5M ammonium acetate, 1-mMEDTA, pH 8.0)
precipitated
100% ethanol
Sequencing of D13S317 alleles
The purified products
Run on ABI 3100 Automated Genetic Analyzer
(Applied Biosystems, Foster City, USA)
Sequencing with HV1 and HV2
regions of mitochondria
PCR product of HV1 and HV2
regions of mitochondria
purified
1U of shrimp alkaline phosphatase
(MBI Fermentas, USA)
37ºC for 15 min
2 U of exonuclease I (NEB, USA)
80ºC for 15 min
The purified products
quantified
electrophoresis on a 2% agarose gel
Sequencing with HV1 and HV2
regions of mitochondria
50-ng DNA of each of the purified products
Big-Dye Terminator
(Applied Biosystems, Foster City, USA) kit
forward primers of HV1 and HV2 mitochondrial regions
PCR products
precipitated
100% ethanol
wash
70% ethanol
Sequencing with HV1 and HV2
regions of mitochondria
The purified products
Run on ABI 3100 Automated Genetic Analyzer
(Applied Biosystems, Foster City, USA)
Results
STR
Table 1 Genotype of the mother and the questioned child and the suspected father
for autosomal STR loci and maternity and paternity indices
Fig.1 Electropherogram of genotypes with peak heights of the mother,
the questioned child and the suspected fatherat test locus D13S317
with internal control locus TPOX (shown in box)
X-Chromosome and HV1 and HV2
regions of mitochondria
Table 2 Genotype of the mother and the questioned child for six chromosome STR loci
The maternal X-chromosome STR alleles and the sequences of
HV1 and HV2 regions of mitochondria in the questioned child
matched with those of the mother.
Sequences of D13S317 locus
TATC
Fig. 3 (A) Forward and reverse sequences of D13S317 locus with flanking regions in the questioned
child. GDB PCR primer sequence is in bold letters and Promega Powerplex 16 kit primers sequence
is underlined . The STR regions are shown in italics. (B) The expansion of microsattellite
repeat motifs in the questioned child is underlined in electropherogram
Table 3 Ratio of peak height of the questioned child,
the mother and the suspected father at D13S317 locus
in relation to the internal homozygote locus (TPOX) as control
Fig.2 Allele frequency distribution at D13S317 locus in Indian population
from which the samples were derived for the study (N=114)
Table 4 Distribution of age mothers and
fathers at the time of conception
Conclusion
The mother and the suspected
father are the biological parents of
the child.
Thank you
for attention…
Question???
Normal replication
5’
3’
CAG CAG
GTC GTC
CAG
GTC
3’
5’
Backward slippage causes insertion
5’
3’
3’
CAG CAG
GTC GTC
GTC
5’
3’
CAG CAG
GTC GTC
CAG
GTC
5’
3’
5’
Forward slippage causes deletion
5’
3’
CAG CAG
GTC GTC
3’
5’
3’
CAG CAG
GTC GTC
3’
5’
5’