molecular marker RFLP, and application

INTRODUCTION
RFLP
Analysis technique
APPLICATION OF RFLP
FORENSIC
DISEASE PROGNOSIS
GENETIC COUNSELING
PEDIGREE
VARIETAL
Advantages of RFLP
Disadvantages of RFLP
CONCLUSIONS
REFERENCES

1. Molecular Marker RFLP By KAUSHAL KUMAR SAHU Assistant Professor (Ad Hoc) Department of Biotechnology Govt. Digvijay Autonomous P. G. College Raj-Nandgaon ( C. G. )

2. Synopsis INTRODUCTION RFLP Analysis technique APPLICATION OF RFLP FORENSIC DISEASE PROGNOSIS GENETIC COUNSELING PEDIGREE VARIETAL Advantages of RFLP Disadvantages of RFLP CONCLUSIONS REFERENCES

3. INTRODUCTION Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA. If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme. The similarity of the patterns generated can be used to differentiate species from one another . First developed in the 1970s, it still has many uses.

4. RFLP RFLP stands for "restriction fragment length polymorphism." The first two words refer to the process of DNA fragmenting with specially applied enzymes. Once this takes place, the DNA strands break up into various lengths. Polymorphism in Greek word means "many shapes." This word references the multiple shapes, or lengths, that can result in a definitive match distinctive to each human being.

5. Analysis technique Extraction The first step is extraction of the DNA from the sample, be it blood, saliva, semen or some other biological sample . 2. Production of Restriction Fragments The purified DNA is then cut into fragments by RESTRICTION ENZYMES.

6. 3. Electrophoresis The restriction fragments have negative charge and can be separated by a technique called GEL ELECTROPHORESIS, which separates the pieces of DNA based on their size. with the smaller fragments migrating farther than the larger fragments.

7. 4. DNA transfer by Southern blotting DNA transfer is called ‘Southern blotting’. In this method, the gel is first denatured in a basic solution and placed in a tray. A porous nylon or nitrocellulose membrane is laid over the gel, and the whole weighted down. All the DNA restriction fragments in the gel transfer as single strands by capillary action to the membrane. All fragments retain the same pattern on the membrane as on the gel.

8. 5. DNA hybridization The membrane with the target DNA is incubated with the DNA probe. Incubation conditions are such that if strands on the membrane are complementary to those of the probe, hybridization will occur and labelled duplexes formed.

9. 6. AUTORADIOGRAPHY An X-ray of the blot provides a picture of the sequencing.

10. Analysis technique

11. APPLICATIONS OF RFLP APPLICATION OF RFLP IN FORENSIC APPLICATION OF RFLP IN DISEASE PROGNOSIS APPLICATION OF RFLP IN GENETIC COUNSELING APPLICATION OF RFLP IN PEDIGREE APPLICATION OF RFLP IN VARIETAL

12. APPLICATION OF RFLP IN FORENSIC RFLP was one of the first applications of DNA analysis to forensic investigation. Any type of organism can be identified by examination of DNA sequences unique to that species. To identify individuals, forensic scientists scan 13 DNA regions, that vary from person to person and use the data to create a DNA profile of that individual. There is an extremely small chance that another person has the same DNA profile for a particular set of 13 regions. Using samples from blood, bone, hair, and other body tissues and products.

13. Some Examples of RFLP Uses for Forensic Identification In criminal cases, this generally involves obtaining samples from crime-scene evidence and a suspect, extracting the DNA, and analyzing it for the presence of a set of specific DNA regions. A series of probes bound to a DNA sample creates a distinctive pattern for an individual. Forensic scientists compare these DNA profiles to determine whether the suspect's sample matches the evidence sample. If the sample profiles don't match, the person did not contribute the DNA at the crime scene. If the patterns match, the suspect may have contributed the evidence sample. determine paternity; that is, who the father of the child really is.

14. APPLICATION OF RFLP IN DISEASE PROGNOSIS RFLP allows an investigator to a potentially defective DNA sequence, it can be used effectively in the diagnosis of diseases in which a specific mutational site. Diagnoses utilizing RFLP linkage analysis can be extended further by comparing genotypes between a known whose status is unknown, the approach is applicable to single gene disorders inherited as autosomal dominant , or x-linked traits . Cloned genes can be modified with specific mutational changes at specific sites by using the technique of site directed mutagenesis.

15. EX. (1) SICKLE CELL ANEMIA

16. APPLICATION OF RFLP IN GENETIC COUNSELING If a particular RFLP is usually associated with a particular genetic disease, then the presence or absence of that RFLP can be used to counsel people about their risk of developing or transmitting the disease.

17. APPLICATION OF RFLP IN PEDIGREE In humans, one mating (one pair of parents) rarely has enough children to give reliable ratios. In that case one can still do pedigree analysis. Modern pedigree analysis is much more sophisticated than anything we can do. The pedigree shows the inheritance of a RFLP marker through three generations in a single family. A total of 8 alleles (numbered to the left of the blots) are present in the family. The RFLPs of each member of the family are placed directly below his (squares) or her (circles) symbol and RFLP numbers.

18. APPLICATION OF RFLP IN VARIETAL RFLP analysis is now a standard, well-tested procedure for estimation of genetic diversity. We have evaluated RFLP markers as tools for assessing genetic variation and determining the relationships among different populations from a wide range of geographical origins and representing Species. RFLP uses in Verify varietal identity and stability.

19. Advantages of RFLPs Highly robust methodology with good transferability between laboratories. Co-dominantly inherited and, as such, can estimate heterozygosity. No sequence information required. Because based on sequence homology, highly recommended for phylogenetic analysis between related species. Well suited for constructing genetic linkage maps .

20. Disadvantages of RFLP s Large amounts of DNA required. Low levels of polymorphism in some species. Need a suitable probe library. Time consuming, especially with single-copy probes. Costly Distribution of probes to collaborating laboratories required.

21. CONCLUSIONS The actual analysis time is lengthy and complicated, but is also less prone to error. RFLP analysis is most common techniques used in a variety of scientific areas, including genetics and forensics. Takes advantage of differences in DNA between individuals that result in different fragments when digested with restriction enzymes.

22. REFERENCES BOOKS Cell and molecular biology by Gerald karp 6 th edition. PDF FAILS ( Date- 14\03\13,Time -08:00 PM ) 18591_ restriction fragment length polymorphism(1). pdf Mol Markers Vol1III RFLPs(1). pdf