DNA fingerprinting…….each DNA profile is unique

Presented at :- G. N. Khalsa Matunga, M-19

Presented by :-C. Shweta( T.Y.I.C Roll no. 4 )

30th June 2015

Contents

History

Structure of DNA

Steps involved

Types

Advantages and disadvantages

Conclusion

References

Introduction

Introduction

A technique used especially for identification by extracting and identifying the base pair pattern of an individual's DNA.

Also known as DNA typing, Genetic fingerprinting, DNA profiling.

History

WHO

WHEN

WHERE

1984

Alec jeffreys

Leicester university

Structure of DNA

Steps involved

Gel electrophoresis

PCR (Polymerase chain reaction)

Digestion of DNA

Sample collection

Sample collection

Digestion

Digestion is performed using Restriction enzymes.

Each of them recognize a short, specific sequence of nucleotide bases.

Nowadays many artificial Restriction enzymes are also used.

PCR (Polymerase chain reaction)

Its purpose is to amplify a lot of double stranded DNA with same size and sequence by enzymatic method and cyclic condition.

Steps

Gel electrophoresis

Method to separate DNA or RNA molecules by size.

Carried out by applying electric field which attracts negatively charged nucleic acid molecules till the end.

Shorter ones move faster.

Types

Restriction Fragment

Length Polymorphism

Amplified Fragment Length

Polymorphism

Random Amplification Polymorphic

DNA

Single Sequence Repeats

Inter Simple Sequence Repeats

Restriction Fragment Length Polymorphism

Analyze difference in homologous sequence that can be detected by the presence of fragments of different length after digestion of DNA samples.

Simple language- study of morphology of DNA from different sources which are digested using restriction enzymes into fragments.

(A) (B)

R.ER.E

Digestion

PCR

RFLP working

Gel electrophoresis

Amplified Fragment Length Polymorphism

•Selective PCR amplification of Restriction fragments from a total digest of genomic DNA.

•Developed by Keygene in 1970's.

AFLP working

EcoRI MseI

Ligate adaptors

PCR

Gel electrophoresis

Random Amplification Polymorphic DNA

To analyse difference between individuals in terms of DNA regions either being or not being amplified in a PCR primed by random oligonucleotides sequence.

It is a type of PCR reaction but the segments of DNA that are amplified are random.

RAPD working

Primers

Primers Primers

Primers

Primers

Primers5’

5’

3’

3’

PCR

Gel electrophoresis

Single Sequence Repeats

Analysis of uniqueness present in the genome of all eukaryotes.

A nucleotide repeat sequence such as (dC-dA)n , (dG-dT)n as many as 50,000 times with 'n' varying from 10-60.

Flanking regions which are highly variable among individuals also can be analyzed.

SSR workingNNNNNNNNN CACACACA NNNNNNNNNNN GTGTGTGTG NNNNNNNNN

NNNNNNNNN GTGTGTGTGTGTGTG NNNNNN CACACAC NNNNNNNNN

SSR

A

B

Gel electrophoresis

Inter Simple Sequence Repeats

Analysis of inter simple sequence repeats is carried out i.e those nucleotides which occurs in between SSR.

No. of nucleotides varies from individual to individual.

ISSR working

NNNNNNNNN CACACACA NNNNNNNNNNN GTGTGTGTG NNNNNNNNN

NNNNNNNNN GTGTGTGTGTGTGTG NNNNNN CACACAC NNNNNNNNN

ISSR

A

B

Gel electrophoresis

Advantages and DisadvantagesAdvantages Disadvantages

RFLP Paternity test, Criminal test, Genetic diseases.

Slow and more tedious process, Required more DNA, Months to accomplish.

AFLP Positional cloning of gene of interest, High reproducibility, Many loci can be analyzed simultaneously.

Complex procedure, Non-identical comigrating bands can cause noise.

RAPD Genetic relation, Genetic diversity, Analyze mixed genome samples, Applicable when limited DNA available, low expense.

Markers are dominant i.e. they can’t distinguish whether DNA sequence amplified is heterozygous or homozygous.

SSR and ISSR Genotyping, Gene profiling, DNA fingerprinting

Point mutation on the primer annealing site can lead to occurrence of null alleles.

Short story Narborough,Leicestershire

2 Rape case with murder took place.

Buckland(14yrs old)

Arrested He was not the criminal Collected killer’s

blood samples and analyze its type n enzyme

Analyzed about 4582 male’s blood sample

10% gave +ve result.Colin pitchfork(27 yrs old)

Refused for test And sent Kelly instead.

Later GUILTY

1st suspect in the world to be identified.

Conclusion

DNA analysis remains the key to linking suspects to biological evidence and to identifying individuals in crimes and disasters.

The establishment of paternity in custody and child support litigation.

DNA profiling is used to diagnose inherited disorders and human diseases.

References

[1] DNA Fingerprinting in Plants and Fungi By Kurt Weising, Hilde Nybom, Markus Pfenninger, Kirsten Wolff, Wieland Meyer[2] Jeffreys AJ, Wilson V and Thein SL Hypervariable ‘ minisatellite ' regions in human DNA. Nature 1985 314: 67-73.[3] SAMPLING TECHNIQUES FOR GENETIC ANALYSIS, Tapir specialist group.[4] DNA Amplification & PCR, New England Biolabs.inc[5] Restriction Fragment Length Polymorphism (RFLP), probe, NCBI.[6] Ovidiu Paun and Peter Schönswetter, Amplified Fragment Length Polymorphism (AFLP) - an invaluable fingerprinting technique for genomic, transcriptomic and epigenetic studies, PMC 2012 Dec 3.[7] Penner GA, Bush A, Wise R, Kim W, Domier L, Kasha K, Laroche A, Scoles G, Molnar SJ, Fedak G., Reproducibility of random amplified polymorphic DNA (RAPD) analysis among laboratories, NCBI.[8] Afaf I. Shehata, Haila A. Al- Ghethar, Ali A. Al- Homaidan, Application of simple sequence repeat (SSR) markers for molecular diversity and heterozygosity analysis in maize inbred lines, Saudi Journal of Biological Sciences Volume 16, Issue 2, October 2009, Pages 57–62, science direct

[9] C.E. McGregor, C.A. Lambert, M.M. Greyling, J.H. Louw, L. Warnich, A comparative assessment of DNA fingerprinting techniques (RAPD, ISSR, AFLP and SSR) in tetraploid potato (Solanum tuberosum L.) germplasm, Volume 113, Issue 2, pp 135-144, Springer link.

[10] How does the polymerase chain reaction work?, abpischools.org.uk.[11] Voytas D., Agarose gel electrophoresis. NCBI.[12] The Discovery of DNA Fingerprinting, DNA forensics, news and databases of DNA.

References

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