Multistep microsatellite mutation in the maternally transmitted locus D13S317: a case of maternal...
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Transcript of Multistep microsatellite mutation in the maternally transmitted locus D13S317: a case of maternal...
Multistep microsatellite mutation in the maternallyMultistep microsatellite mutation in the maternally transmitted locus D13S317:transmitted locus D13S317:
a case a case of maternal allele mismatch in the childof maternal allele mismatch in the child
Devinder Singh Negi . Mahfooz Alam . Devinder Singh Negi . Mahfooz Alam . S. Annapurna Bhavani . Javaregowda Nagaraju S. Annapurna Bhavani . Javaregowda Nagaraju
Present by…Present by… Nonglak SinkhanNonglak Sinkhan ID 51312307ID 51312307
Introduction
MicrosatelliteMicrosatellite หรื�อหรื�อShort Tandem Repeats (STR)Short Tandem Repeats (STR)
Y-Chromosomal DNA X-Chromosomal DNA
Mitochondrial DNA
Microsatellite mutation
Purpose
Sample
Blood samples at the Laboratory of DN A Fingerprinting Services of Centre for
DNA Fingerprinting and Diagnostics, H yderabad, India
Materials
Method
Phenol/choroform
DNA samples
DNA samples
Extraction The quantity of DNA
STR AmpFlSTR Profiler Plus, Identifiler (Applied Biosystems, Foster City, USA) PowerPlex (Promega Corporation, Wisconsin, USA)
X chromosome published primer sequences
Mi tochondrial DNA hypervariable regions HV1 and2HV
published primer sequences
AmplificationAmplification
AmplificationAmplification50-ng DNA
200-µM dNTPs
6 pmol primer
2.5- U AmpliTaqGold
1xPCR buffer
1.5-mMMgCl2
ABIGeneAmpPCRsystem9 7 0 0 (Perkin Elmer,N orwalkCT)
Thermal cycling condition :
Denaturation 90ºC ถึ�ง 95ºC
Annealing 50ºC ถึ�ง 55 ºC
Extension 70 °C ถึ�ง 75 ºC
PCR
GeneScan and genotyping
ABI PRISM® 3100 Automated Genetic Analyzer (Applied Biosystems, Foster City, USA)
Sequencing of D13S317 alleles
DNA samples of the suspiciousfather, mother a nd questioned child
PCR allelic product of D13S317 locus
6% polyacrylamide gel electrophoresis (PAGE)
purified
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 Analy zer
(Applied Biosystems, Foster City, USA)
Sequencing with HV1 and HV2 regions of mitochondria
PCR product of HV1 and HV2 regions of mitochondria
purified1 U of shrimp alkaline phosphatase
(MBI Fermentas, USA)
37ºC for 15 min
2 U of exonuclease I (NEB, USA)
80º C for 15 min
The purified products
electrophoresis on a2 % agarose gel
quantified
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) ki
t
forward primers of HV1 and HV2 mitochondrial regions
PCR products
precipitated
100 % ethanol
70 % ethanol
wash
Sequencing with HV1 and HV2 regions of mitochondria
The purified products
Run on ABI 3100 Automated Genetic Analyzer (Applied Biosystems, Foster City, USA)
The purified products
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.
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 sequenceis underlined . The STR regions are shown in italics. (B) The expansion of microsattellite repeat motifs in the questioned child is underlined in electropherogram
TATC
Sequences of D13S317 locus
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
T he mother and the suspected f ather are the biological parents
of the child.
Thank you for
attention…
Question???
Normal replication
CAG CAG CAG
GTC
GTC
CAG
CAG
GTC
CAGGTC
GTCCAG
GTCCAG
GTCCAG
GTCCAG
GTC
CAG
GTCCAG
GTCCAG
GTC
GTC
CAG
GTC
GTC5’
5’
5’
5’
5’
5’
5’
5’
5’
5’3’
3’
3’
3’
3’
3’
3’
3’
3’
3’
Backward slippage causes insertion
Forward slippage causes deletion