1. 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

2. Introduction

3. Microsatellite หรือ Short Tandem Repeats (STR)

4. Y-Chromosomal DNA
X-Chromosomal DNA

5. Mitochondrial DNA

6. Microsatellite mutation

7. Purpose

8. Materials
Sample
Blood samples at the Laboratory of DNA Fingerprinting Services of Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India

9. Method

10. Extraction The quantity of DNA
DNA samples
Phenol/choroform
DNA samples

11. 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

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

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

14. GeneScan and genotyping
ABI PRISM® 3100 Automated Genetic Analyzer
(Applied Biosystems, Foster City, USA)

15. 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

16. Sequencing of D13S317 alleles
The purified products
Run on ABI 3100 Automated Genetic Analyzer
(Applied Biosystems, Foster City, USA)

17. 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

18. 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

19. 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)

20. Results

21. STR
Table 1 Genotype of the mother and the questioned child and the suspected father
for autosomal STR loci and maternity and paternity indices

22. 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)

23. 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.

24. 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

25. 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

26. 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)

27. Table 4 Distribution of age mothers and
fathers at the time of conception

28. Conclusion
The mother and the suspected father are the biological parents of the child.

29. Thank you
for attention…

30. Question???

31. 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