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DNA fingerprinting 1
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 )
DNA fingerprinting
Contents
Introduction
History
Steps involved
Types
Advantages and disadvantages
Conclusion
References
Structure of DNA
2
DNA fingerprinting
Structure of DNA
3
DNA fingerprinting
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.
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DNA fingerprinting
History
WHO
WHEN
WHERE
1984
Alec jeffreys
Leicester university
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DNA fingerprinting
Steps involved
Southern blotting
Gel electrophoresis
PCR (Polymerase chain reaction)
Digestion of DNA
Sample collection
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DNA fingerprinting
Sample collection
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DNA fingerprinting 8
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.
DNA fingerprinting 9
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.
DNA fingerprinting
Steps
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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.
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DNA fingerprinting 13
SOUTHERN BLOTTING A procedure allowing DNA in
electrophoresis gel to be transferred to nitrocellulose membrane.
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Types
Restriction Fragment
Length Polymorphism
Amplified Fragment Length
Polymorphism
Random Amplification Polymorphic
DNA
Single Sequence Repeats
Inter Simple Sequence Repeats
DNA fingerprinting 15
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.
DNA fingerprinting
(A) (B)
R.ER.E
Digestion
PCR
RFLP working
Gel electrophoresisSouthern blotting
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DNA fingerprinting 17
Amplified Fragment Length Polymorphism
•Selective PCR amplification of Restriction fragments from a total digest of genomic DNA.
•Developed by Keygene in 1970's.
DNA fingerprinting
AFLP working
EcoRI MseI
Ligate adaptors
PCR
Gel electrophoresis
Southern blotting
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DNA fingerprinting 19
Random Amplification Polymorphic DNA
To analyze 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.
DNA fingerprinting
RAPD working
Primers
Primers Primers
Primers
Primers
Primers5’
5’
3’
3’
PCR
Gel electrophoresis
Southern blotting
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DNA fingerprinting 21
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.
DNA fingerprinting
SSR workingNNNNNNNNN CACACACA NNNNNNNNNNN GTGTGTGTG NNNNNNNNN
NNNNNNNNN GTGTGTGTGTGTGTG NNNNNN CACACAC NNNNNNNNN
SSR
A
B
Gel electrophoresis Southern blotting
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DNA fingerprinting 23
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.
DNA fingerprinting
ISSR workingNNNNNNNNN CACACACA NNNNNNNNNNN GTGTGTGTG NNNNNNNNN
NNNNNNNNN GTGTGTGTGTGTGTG NNNNNN CACACAC NNNNNNNNN
ISSR
A
B
Gel electrophoresis Southern blotting
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DNA fingerprinting
Advantages and DisadvantagesAdvantages Disadvantages
RFLP Paternity test, Criminal test, Genetic diseases.
Slow and more tedious process, Required more DNA.
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.
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DNA fingerprinting
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. 26
DNA fingerprinting 27
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.
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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
DNA fingerprinting
[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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DNA fingerprinting
THANK YOU
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