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 หรือ 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

Amplification 50-ng DNA 200-µM dNTPs 6 pmol primer 2.5-U AmpliTaq Gold 1xPCR buffer 1.5-mMMgCl2

Thermal cycling condition : PCR Thermal cycling condition : Denaturation 90ºC ถึง 95ºC Annealing 50ºC ถึง 55 ºC Extension 70 °C ถึง 75 ºC ABI GeneAmp PCR system 9700 (Perkin Elmer,Norwalk CT)

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 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

from which the samples were derived for the study (N=114) 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???

Backward slippage causes insertion Normal replication 5’ 3’ CAG CAG CAG 3’ 5’ GTC GTC GTC Backward slippage causes insertion 5’ CAG 3’ CAG CAG 5’ 3’ GTC GTC GTC 5’ CAG 3’ CAG CAG CAG 3’ 5’ GTC GTC GTC Forward slippage causes deletion 5’ 3’ CAG CAG 5’ 3’ GTC GTC GTC 5’ 3’ CAG CAG 5’ 3’ GTC GTC GTC