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