DNA Fingerprinting

By : Marvi Altaf & Group
Biology Department
Preston University Islamabad

1. DNA Fingerprinting …….each DNA profile is unique Presented by :- Marvi Altaf Amna Liaqat Afshan Gohar Mehwis h Khalid Shumaila Raza Namal Khan

2. Contents I ntroduction 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. What is DNA Profiling? A technique used by scientists to distinguish between individuals of the same species using only samples of their DNA. The only difference between people (or any animal) is the order of the base pairs . The information contained in DNA is determined primarily by the sequence of letters along the zipper. The different sequences is the same as the word "POST" has a different meaning from "STOP" or "POTS," even though they use the same letters

6. History Who When Where 1984 Alec jeffreys Leicester university DNA fingerprinting 6

7. It was first used in the law courts of England in 1987 to convict a man in a rape case. It has now been used successfully in many crime and paternity cases in worldwide . Although 99.9% of human DNA sequences are the same in every person, enough of the DNA is different to distinguish one individual from another, unless they are monozygotic twins.  DNA profiling uses repetitive sequences that are highly variable, called variable number tandem repeats (VNTRs), particularly short tandem repeats (STRs) . VNTR loci are very similar between closely related humans. The analysis of variable number of tandem repeats (VNTRs), to detect the degree of relatedness to another sequence of oligonucleotides , making them ideal for DNA fingerprinting.

8. S teps involved DNA fingerprinting 8

9. DNA FINGERPRINTING ( DNA PROFILING ) Cells broken down to release DNA DNA strands cut into fragments Fragments separated Pattern of fragments analysed

10. Overall Process http://www.youtube.com/watch?v=drC7rR7CIBg Video of DNA Fingerprinting Process

11. Sample collection DNA fingerprinting 11

12. Isolation of the DNA! In order for a DNA fingerprint to be made you must first gather DNA from the person you are fingerprinting. There are many things you could use to gather DNA… Blood Hair Skin Saliva Or any other cell with DNA in it. Human Cheek Cells

13. Restriction Enzymes : 1) Used to cut DNA into pieces that are specific to each individual persons DNA. Specific Enzymes cut the DNA into pieces at specific places as well. For Example… EcoR1 cuts only when the sequence GAATTC occurs.

14. The sections of DNA that are cut out are called restriction fragments. This yields thousands of restriction fragments of all different sizes because the base sequences being cut may be far apart (long fragment) or close together (short fragment).

15. 2 ) Transfer of DNA to nylon: The distribution of DNA pieces is transferred to a nylon sheet by placing the sheet on the gel and soaking them overnight. 3) Probing :  Adding radioactive or colored probes to the nylon sheet produces a pattern called the DNA fingerprint. Each probe typically sticks in only one or two specific places on the nylon sheet. 4) DNA fingerprint : The final DNA fingerprint is built by using several probes (5-10 or more) simultaneously. It resembles the bar codes used by grocery store scanners

16. Types of DNA fingerprinting methods are… Electrophoresis. Polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP). Random Amplified Polymorphic DNA(RAPD). Amplified fragment length polymorphism (AFLP).

17. 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 18

18. Electrophoresis… Electrophoresis is a separations technique that is based on the the mobility of ions in an electric field. Positively charged ions migrate towards a negative electrode and negatively-charged ions migrate toward a positive electrode. Ions have different migration rates depending on their total charge, size, and shape, and can therefore be separated.

19. 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 20

20. The polymerase chain reaction (PCR) was developed by Karry Mullis of the Cetus Corporation in 1983 . In this process, the DNA sample is denatured into the separate individual strands. Specific DNA primers are used to hybridize to two corresponding nearby sites on opposite DNA strands in such a fashion that the normal enzymatic extension of the active terminal of each primer (that is, the 3’ end) leads toward the other primer. In this fashion, two new copies of the sequence of interest are generated.

21. Repeated denaturation, hybridization, and extension in this fashion produce an exponentially growing number of copies of the DNA of interest. The PCR analysis amplified isolated regions on the strands of the DNA under examination. 

22. SOUTHERN BLOTTING A procedure allowing DNA in electrophoresis gel to be transferred to nitrocellulose membrane. DNA fingerprinting 23

23. Types DNA fingerprinting 24

24. 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 25

25. RFLP analyzes the length of the strands of the DNA molecules with repeating base pair patterns. The basic technique for detecting RFLPs involves fragmenting a sample of DNA by a restriction enzyme, which can recognize and cut DNA wherever a specific short sequence occurs, in a process known as a restriction digest. The resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis, and transferred to a membrane via the Southern blot procedure. 

26. Random Amplified Polymorphic DNA…  It is a type of PCR reaction, but the segments of DNA that are amplified at random. RAPD creates several arbitrary, short primers (8–12 nucleotides), then proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify. By resolving the resulting patterns, a semi-unique profile can be gleaned from a RAPD reaction. RAPD does not require any specific knowledge of the DNA sequence of the target organism: the identical 10-mer primers will or will not amplify a segment of DNA, depending on positions that are complementary to the primers' sequence.

27. Amplified fragment length polymorphism …  This technique was also faster than RFLP analysis and used PCR to amplify DNA samples. It relied on variable number tandem repeat (VNTR) polymorphisms to distinguish various alleles, which were separated on a  polyacrylamide gel. By using the PCR analysis to amplify the minisatellite loci of the human cell, this method proved quicker in recovery than the RFLP. However, due to the use of gel in its analysis phase, there are issues of bunching of the VTRN's, causing misidentifications in the process. 

28. 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 29

29. SSR working NNNNNNNN CACACA NNNNNNNNN NNNNNNNN NNNNNNN GTGTGTGTGTGTG NNNNN CACACA NNNNNNNN SSR A B Gel electrophoresis Southern blotting DNA fingerprinting 30 GTGTGTGT

30. 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 31

31. ISSR working NNNNNNNN CACACA NNNNNNNNN GTGTGTGT NNNNNNNN NNNNNNNN GTGTGTGTGTGTG NNNNN CACACA NNNNNNNN ISSR A B Gel electrophoresis Southern blotting DNA fingerprinting 32

32. 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 33

33. Applications… 1) Diagnosis and Developing cures for inherited disorders : DNA fingerprinting is used to diagnose inherited disorders in both prenatal and newborn babies in hospitals around the world. These disorders may include cystic fibrosis, hemophilia, Huntington's disease, familial Alzheimer's, sickle cell anemia, thalassemia , and many others. Early detection of such disorders enables the medical staff to prepare themselves and the parents for proper treatment of the child. In some programs, genetic counselors use DNA fingerprint information to help prospective parents understand the risk of having an affected child.

34. In other programs, prospective parents use DNA fingerprint information in their decisions concerning affected pregnancies. 2)Biological Evidence to Identify Criminals: Where fingerprints are not available but biological specimens are available like blood or semen stains, hair, or items of clothing at the scene of the crime then these items may prove to be valuable sources of DNA of the criminal. Since the year 1987, innumerable cases have been solved with the help of DNA fingerprint evidence.

35. ( DNA PROFILING ) Uses Of DNA Fingerprinting: Medical researchers, forensic biologists, genealogists and anthropologists all use DNA fingerprinting to identify individuals or to determine the relation between individuals or groups of individuals. Information gained from DNA fingerprinting can answer questions concerning the structure and migration of human populations, establish paternity and place a suspect at the scene of a crime.

36. ( DNA PROFILING ) Benefits and limits of DNA Fingerprinting: Benefits of DNA Fingerprinting:   The most important benefit of DNA fingerprinting is that there is strong similarities shown between genetic fingerprints of parents and children.   This is a benefit because a child's genetic fingerprint is made up of half the father's genetic information and half of the mother's information.  This means that the bands of a child's genetic fingerprint will match the bands on both of their parents, making it possible to establish paternity and maternity tests. 

37. Limitations of DNA Fingerprinting: An uncontaminated DNA blood sample taken from a crime scene. One of the main problems with the process of DNA fingerprinting is that the sample can be easily ruined.  The tiniest pieces of genetic junk can contaminate DNA samples, causing them to be useless.  Although DNA fingerprinting requires a good sample to work with, this problem can be solved by using the newer technique called PCR.  PCR can use extremely small samples of DNA and produce a much faster result.  But this also means the DNA samples that PCR uses are even more likely to be contaminated because of their size, as it is harder to find a small sample with hardly any contamination.  Another limitation of fingerprinting is that the procedure is so complex and hard to read the DNA patterns, that sometimes the juror finds the evidence almost invisible.  

38. ( DNA PROFILING ) DNA Fingerprinting In Plants: The basic methodology of DNA profiling in plants involve first the extraction of DNA from plant cells, quantification and quality assessment of extract. The further steps are of two types,   1) PCR based.  -   RAPD, ISSR, SSR  2) Non PCR based. – RFLP. The in the PCR based techniques diluted DNA is mixed with a master mix comprising the PCR buffer, DNTPS, primer, water and the Taq polymerase enzyme in a PCR eppendorf tube .The mixture is loaded into the PCR. The PCR is pre-programmed for appropriate number of cycles and temperature variations depending on the technique.  After required cycles, the samples are subjected to electrophoresis, either AGE or PAGE , depending on the technique. The staining is done for revealing the banding pattern.

39. Conclusion: With a beginning as a mere forensic tool, the world of DNA fingerprinting has a gone a long way in revealing the genetic identity of living beings. With massive evolution in concepts and techniques it has given a lot to various fields cutting across the spectrum. In plants it has not only helped in identifying species but also in defining a new realm in plant genomics, plant breeding and in conserving the biodiversity. With world paving way for developments in biotechnology, DNA fingerprinting promises a very powerful tool in our future endeavors.  

40. 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 41

41. R eferences [ 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 42