RFLP s with VNTR analysis

Transcription

1 RFLP s with VNTR analysis

2 The most powerful and awesome tool acquired by humans since the splitting of atoms The Time Magazine (U.S.A)

3 INTRODUCTION DNA profiling (also called DNA testing, DNA typing, or genetic fingerprinting) is technology of using tandem repeats of individuals to identify individuals

4 ALTHOUGH THE MAJORITY OF OUR DNA IS THE SAME, THERE ARE SHORT PIECES CALLED MICRO- SATELLITES, WHICH REPEATS MANY TIMES IN A PERSON S DNA. IT S THE PATTERN OF REPEATS THAT Knowing these micro-satellite DNA sequences is the basis of DNA PROFILING ARE DIFFERENT BETWEEN INDIVIDUALS.

5 KEY CONCEPT DNA profiling or finger printing identifies people at molecular level.

6 DNA fingerprints are based on parts of an individual s DNA that can by used for identification. Based on non coding regions of DNA Non coding regions have repeating DNA sequences Number of repeats differs between people Banding pattern on a gel is a DNA fingerprint

7 TYPES OF DNA FINGERPRINTING

8 RFLP-based (Restriction fragments length polymorphism) 1 st step is the Extraction of DNA DNA can be extracted from almost any human tissue. Sources of DNA found at a crime scene might include blood, semen, tissue from a deceased victim, cells in a hair follicle, and even saliva. DNA extracted from items of evidence is compared to DNA extracted from reference samples from known individuals, normally from blood.

9 DNA EXTRACTION METHOD To extract DNA, most commonly used detergent is Sodium Dodecyl Sulfate (SDS).

10 Digestion of proteins and lipids around the cell and it nucleus

11 DNA is extracted from sample DNA in the nucleus of the cell is molded, folded, and protected by proteins Proteinase K is used to break up the proteins

12 Ethanol Precipitation The DNA must be purified from the cell extract. Ethanol lowers the effective water concentration, causing large bio-molecules to interpenetrate and aggregate. The result is a visible precipitate at the interface, where the ethanol is concentrated. DNA will rise into the alcohol layer from the cell extract layer. The protein and grease parts dissolve in the bottom, watery layer

13 Step 2.DNA cuts with the help of Restriction Endonuclease DNA is cut into millions of small fragments with the help of restriction Endonuclease Restriction enzymes chop DNA at specific sequences. the patterns occur in different places in different individual, the length of the fragment differs from person to person.

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15 We are ALL a little bit different! BIG Brenna s DNA LITTLE Leila s DNA

16 Our DNA has different sizes of pieces so it makes a different pattern when it s all cut up Brenna s DNA Leila s DNA

17 RFLP Analysis Everyone has genetic sequences called variable number tandem repeats, or VNTRs Everyone has different amounts of VNTRs The VNTRs make the different sized RFLPs

18 GEL ELECTROPHORESIS Fragments of DNA from restriction enzyme cleavage are separated from each other when they migrate through a support called an agarose gel It is similar to the yummy food Jell-O gelatin It is actually made out of some of the same ingredients The size-based separation of Molecules of DNA separate based on size when an electric current is applied to an agarose gel This is gel electrophoresis

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20 GEL ELECTROPHORESIS The separated DNA fragments are then drawn out of the gel using a nylon membrane The nylon membrane is treated with chemicals that break the hydrogen bonds in DNA and separate the strands The single stranded DNA is cross linked to the nylon membrane The nylon membrane is incubated with a radioactive probe of single stranded DNA complementary to the VNTRs

21 DNA, having repeat sequences, will only be bound to the radioactive or fluorescent probe.

22 The final step to making a genetic fingerprint is to place a photographic film on top of the nylon surface. The probes leave marks on the film wherever they attached to the RFLPs. Dark bands will then show up when the film is developed, which marks the length of the RFLPs that were hybridized. Researchers are then able to read the fingerprint and match it to others. They do this by placing the xray on a light background, and comparing the RFLP lengths in the DNA from the crime scene, to the DNA of the suspect.

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24 DNA fingerprints can be used to determine which bone fragment belongs to which individual.

25 STRs & PCR Currently, the most popular method of DNA fingerprinting are short tandem repeats, or STRs for short Unlike VNTRs which analyze minisatellites that have repeat sequences of 9-80 base pairs, STRs use microsatellites which have repeat sequences of only 2-5 base pairs, introducing the less is more philosophy to the world of DNA fingerprinting. This was a big step forward in forensic science since the length of DNA fragment being analyzed is short enough to be amplified by polymerase chain reaction (PCR), so now we are able to analyze a very small sample of DNA that is quicker and easier

26 PCR was developed in the mid 1980 s and used the same principles that cells use to replicate DNA to amplify the specified region, which is usually between 150-3,000 base pairs in length. STEPS IN PCR OPERATION The solution is heated to 95 C to unzip the double helix DNA structure. The solution is cooled to 60 C to allow the primers to bind to the ends of the DNA. The solution is then reheated to 75 C which is the optimal temperature for the Taq polymerase to create new copies of each DNA strand

27 ADVANTAGES OF STRs OVER RFLP/VNTR STRs are currently the most popular type of DNA fingerprint, since the whole PCR process takes only a few hours, compared to RFLP/VNTR probe hybridization and film exposure which can take several days. STRs can use much smaller samples of DNA than RFLPs/VNTRs, and can even use partially degraded DNA to create a fingerprint Thus, the integrity and quality of the DNA sample is not as great a factor with STRs than with the traditional methods of DNA fingerprinting

28 DISADVANTAGE OF STRs The only disadvantage of the STR approach is it is sensitive to contaminating DNA, so usually the STR approach is used first, followed by a VNTR analysis if contamination is suspected, and enough DNA is available.

29 Practical Applications of DNA Paternity and Maternity cases Parent-child VNTR pattern analysis has been used to solve standard fatheridentification cases Fingerprinting

30 Criminal Identification and Forensics DNA isolated from blood, hair, skin cells, or other genetic evidence left at the scene of a crime can be compared

31 Diagnosis of Inherited Disorders Diagnose inherited disorders in both prenatal and newborn babies These disorders may include cystic fibrosis, hemophilia, Huntington's disease, familial Alzheimer's, sickle cell anemia, thalassemia, and many others.

32 CONCLUSION DNA fingerprinting is the most sophisticated way to identify living organisms. DNA cannot easily be altered once it is left at a crime scene or deposited with a mummy, which makes it a strong forensic tool. RFLPs and VNTRs are the traditional methods of fingerprinting DNA, which uses a relatively large sample that uses the method of probe hybridization to detect polymorphisms in the DNA. STRs are the most current form of DNA fingerprinting, which is PCR based and uses a very small sample of DNA.