Dna fingerprinting
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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 1
- 2. Contents Introduction History Steps involved Types Advantages and disadvantages Conclusion References Structure of DNA DNA fingerprinting 2
- 3. Structure of DNA DNA fingerprinting 3
- 4. 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. DNA fingerprinting 4
- 5. History 1984 Alec jeffreys Leicester university DNA fingerprinting 5
- 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 8
- 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 9
- 11. 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. DNA fingerprinting 11
- 13. SOUTHERN BLOTTING A procedure allowing DNA in electrophoresis gel to be transferred to nitrocellulose membrane. DNA fingerprinting 13
- 14. Types Restriction Fragment Length Polymorphism Amplified Fragment Length Polymorphism Random Amplification Polymorphic DNA Single Sequence Repeats Inter Simple Sequence Repeats DNA fingerprinting 14
- 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 15
- 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 17
- 18. AFLP working EcoRI MseI Ligate adaptors PCR Gel electrophoresisDNA fingerprinting 18
- 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 19
- 20. RAPD working Primers Primers Primers Primers Primers Primers 5’ 5’ 3’ 3’ PCR Gel electrophoresis DNA fingerprinting 20
- 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 21
- 22. SSR working NNNNNNNNN CACACACA NNNNNNNNNNN GTGTGTGTG NNNNNNNNN NNNNNNNNN GTGTGTGTGTGTGTG NNNNNN CACACAC NNNNNNNNN SSR A B Gel electrophoresis DNA fingerprinting 22
- 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 23
- 24. ISSR working NNNNNNNNN CACACACA NNNNNNNNNNN GTGTGTGTG NNNNNNNNN NNNNNNNNN GTGTGTGTGTGTGTG NNNNNN CACACAC NNNNNNNNN ISSR A B Gel electrophoresis DNA fingerprinting 24
- 25. Advantages and Disadvantages Advantages 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. DNA fingerprinting 25
- 26. 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. GUILTY 1st suspect in the world to be identified. DNA fingerprinting 26
- 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. DNA fingerprinting 27
- 28. 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 28
- 29. [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 DNA fingerprinting 29
Editor's Notes
- A vey good morning to one and all, myself Shweta chavan. My topic for the presentation is DNA fingerprinting… which says each DNA profile is unique.
- Points I am going to cover about DNA fingerprinting are structure of DNA, introduction, history, Steps involved, types, advantages and disadvantages, conclusion.
- Before we move further first let us know what is DNA , DNA is an extremely long macromolecule which is the main component of chromosome. It carries genetic information of an organism. As we can see it is double stranded twisted ladder like structure containing nitrogen bases that are purines, pyrimidines, phosphate moiety which gives the dna negative charge, and ribose sugar. There are majorly two bonds, between the bases there is weak hydrogen bond whereas the strong covalent bond is seen between phosphate backbone i.e. phosphodiester bond
- DNA fingerprinting is a different set of features that are present inside DNA, different sequences, uniqueness of the genome, found in only one individual. Suppose you are having a typical type n structure or sequence of DNA and I m having something unique than it is called as fingerprinting. As we all know what do you mean by normal fingerprinting, no human on this planet have same fingerprint likewise no two people share same type of DNA, not even between individual belonging to the same family. By the proper definition DNA fingerprinting is said to be a technique used for identification by extracting and identifying the base pair pattern of an individual’s DNA. It is also called as DNA typing, Genetic fingerprinting, DNA profiling.
- Any idea who invented this technique? The term was tossed by Alec jeffreys in the year 1984 at Leicester university. He is a British geneticist, and also a professor at the same university.
- DNA fingerprinting involves following steps, sample collection, Digestion of DNA using restriction enzyme. PCR for multiplying the no.of strands, gel electrophoresis for running the sample across the electric current, southern blotting for analyzing it further or simply printing it on a nylon membrane. We shall see these points in detail in next slides.
- Sample from which we can extract DNA for analysis can be Drop or traces of Blood, fragments or Root of hair, Skin scrapping or in some cases skin found under the nails while a fight also Saliva and Semen can also be used.
- Digestion here means cutting the genome or long isolated DNA fragment into small fragments. These RE are also called as molecular scissors as they are very specific with site of cleavage. Nowadays many artificial restriction enzymes are also designed to cut desired site. E.g. EcoR1, Mse1 etc. DNA is cut down so that it can be separated easily.
- PCR is used for multiplying the digested DNA strands. Main principle is to obtain dsDNA strands of same size, sequence by using specific conditions.
- Mechanism of pcr is the dsDNA is denatured that is the temperature is so kept that the double helical strand is open, once the strand is open the primers ae annealed to the ends and the extension begins. This whole process is called as one cycle. Depending on the amount of DNA one need the no.of cycles ae set accordingly.
- The name itself describe the technique as in it is something to do with electric current. The isolated and treated samples with dye are loaded on an agarose gel and the gel is allowed to diffuse electric current across it. As we know that DNA is negatively charged they will migrate from towards the positive end of the gel. Shorter ones move faster because of their size as well as pore size of the gel.
- These are equipment of gel electrophoresis i.e. casting plate to cast gel, comb for making wells, power supply for separation.
- Next we preform southern blot. An apparatus is set to transfer the DNA into gel on to a solid support. The gel is placed on a sponge soaked into alkaline solution. Nitrocellulose paper is placed on top of the gel and a stack of paper towel is placed on top of nitrocellulose paper. The solution is drawn up the stack and the DNA denatures in the solution as it is carried up the stack. The dna stops traveling when it reaches the nitrocellulose paper. If you see the paper you will see that it resembles the same bands on the gel. Further for more specific detection radio labelled molecules are used.
- Now as we know the basics and principle of DNA fingerprinting we shall see its various types. Commonly called as rflp, aflp, rapd, ssr and issr.
- In this type the DNA is digested using the same restriction enzyme. If the site of cleavage in any of the sample is same than we can interpret that the DNA of the two sample matches in some characters. By the proper definition RFLP can be described as technique which can analyse difference in homologous sequence that can be detected by the presence of fragments of different length after digestion of DNA samples. Its working can be seen as following.
- 2nd technique is AFLP, in this we amplify a particular sequence of desired DNA and than study it accordingly. It was first developed by Keygene in 1970’s. Its working is as follows.
- Here the primers are allowed to ligate the genome randomly and are amplified using PCR. It is a type of PCR reaction but the segments of DNA that are amplified are random. Its working is as follows.
- Single sequence repeats are those in which we have CA and GT sequence repeating n times. This n varies from individual to individual hence leading to the uniqueness of the DNA. Its working is as follows.
- The region between the single sequence repeats like CA and GT also varies like it does with single sequence repeats. It works in following way.
- Advantages 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.
- This is short story how the dna fingerprinting came into existence. In a small city narborough at licestershire 2 rape cases with murder took place an individual named buckland was arrested according to alec he was not the criminal so he started his own investigation, in that he analyzed about 4582 males blood sample out of these 10% of them gave positive result. But one of the male in the city named colin pitchfork refused for the test and sent his companion kelly instead. Later in a club when he was describing the scene to one of his friend he got caught at the end. So colin pitchfork was the first suspect in the world to be identified.