VNTR and RFLP

1. Contents: RFLP What is RFLP… Method of DNA analysis by RFLP Applications of RFLP VNTR What is VNTR… VNTR Structure and allelic variation Use of VNTR in analyzing genetics VNTR inheritance General method of VNTR Applications of VNTR

2. RFLP is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples. What is RFLP…

3. Most RFLP markers are co-dominant (both alleles in heterozygous sample will be detected) and highly locus-specific. An RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis. What is RFLP…

4. The method of analysis of DNA by RFLP involves the following steps: 1- In the first step fragmentation of a sample of DNA is done by a restriction enzyme, which can recognize and cut DNA wherever a specific short sequence occurs, in a process known as a restriction digest. Method of DNA analysis by RFLP

5. 2- The resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis. 3- Then transferred to a membrane via the Southern blot procedure. 4- Hybridization of the membrane to a labeled DNA probe will done and then determines the length of the fragments which are complementary to the probe.

6. 5- Then we will observe the fragments of different length. An RFLP occurs when the length of a detected fragment varies between individuals. Each fragment length is considered an allele, and can be used in genetic analysis.

7. RFLPs can be used in many different settings to accomplish different objectives as: 1- Paternity case: RFLPs can be used in paternity cases or criminal cases to determine the source of a DNA sample. (i.e. it has forensic applications). Applications of RFLP

8. 2- Detection of Recombination rate: RFLPs can be used to measure recombination rates which can lead to a genetic map with the distance between RFLP loci. 3- Agriculture: Direct method for selecting desirable genes such as disease resistance

9. 4- Genetic mapping: Determine disease status of an individual, i.e. Huntington’s chorea Cystic fibrosis Sickle cell anemia 5- Genetic Counseling: Very important when discussing results with patients or parents who use this technology to have children who are free of genetic disease.

10. 6- Disease detection: RFLPs can be used determine the disease status of an individual. (e.g. it can be used in the detection of particular mutations)

11. What is VNTR… A  Variable Number Tandem Repeat  (or  VNTR ) is a location in a genome where a short nucleotide sequence is organized as a tandem repeat. These can be found on many chromosomes, and often show variations in length between individuals. Each variant acts as an inherited allele, allowing them to be used for personal or parental identification. The size of repeat is about 7 to few tens of base pairs long.

12. VNTR Structure and allelic variation The rectangular blocks represent each of the repeated DNA sequences at a particular VNTR location. The repeats are tandem - they are clustered together and oriented in the same direction. Individual repeats can be removed from (or added to) the VNTR via recombination or replication errors. The VNTR blocks can be extracted with restriction enzymes and analyzed by RFLP, or amplified by the polymerase chain reaction (PCR) technique and their size determined by gel electrophoresis.

13. Use of VNTR in analyzing genetics VNTRs were an important source of RFLP genetic markers used in linkage analysis (mapping) of genomes. When removed from surrounding DNA by the PCR or RFLP methods, and their size determined by gel electrophoresis or Southern blotting, they produce a pattern of bands unique to each individual. When tested with a group of independent VNTR markers, the likelihood of two unrelated individuals having the same allelic pattern.

14. VNTR inheritance In analyzing VNTR data, two basic genetic principles can be used: Identity Matching- both VNTR alleles from a specific location must match. If two samples are from the same individual, they must show the same allele pattern. Inheritance Matching- the VNTR alleles must follow the rules of inheritance. In matching an individual with his parents or children, a person must have an allele that matches one from each parent. If the relationship is more distant, such as a grandparent or sibling, then matches must be consistent with the degree of relatedness.

15. General method of VNTR In first step we use restriction enzymes to cut the VNTR blocks from its specific sides and analyzed by RFLP In second step we amplified PCR techniques In last step we run it on gel electrophoresis which also determined its length

16. Applications of VNTR Microbiology DNA fingerprinting Genetic diversity Biology research Forensics Mapping of genomes Breeding patterns in populations of wild or domesticated animals