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Dna chips, RFLPs & dna fingerprint

  1. Microarrays (DNA Chips) Tapeshwar Yadav (Lecturer) BMLT, DNHE, M.Sc. Medical Biochemistry
  2. Microarrays (DNA Chips) • These are recent advancements for DNA sequencing as result of advances made in automation miniarization. • A large no. of DNA probes, each one with difference sequence, are immobilized at defined positions on the solid surface, made up of either nylon or glass.
  3. Applications of DNA chips  There have been many successes with this relatively new technology of DNA chips. Some of them are:- I. Identification of genes responsible for the development of nervous system. II. Detection of genes responsible for inflammatory diseases. III. Construction of microarrays for gene in the genome of E.coli and almost all the genes of the yeast saccharomyces cerevisiae.
  4. Contd… IV. Detection and screening of single nucleotide polymorphisms. V. Rapid detection of microorganisms for environmental monitoring. VI. Expression of several genes in prokaryotes has been identified.
  5. Applications 1. Functional genomics In the functional genomics area, DNA chips are used for measurement of expression level of genes or expression patterns of genes. A limited number of genes involved in a pathway may be selected. 2. Diagnostics and genetic mapping DNA chips are used for diagnositcs. For example, diagnostic chips are prepared to detect mutant alleles in cystic fibrosis and beta globin genes. DNA chips are also used for genotyping of hepatitis C virus in blood samples.
  6. 3. DNA sequencing by hybridization (SBH) DNA sequence is determined by using DNA chips. These DNA chips contain set of oligonucleotides of particular size. Hybridization is carried out with DNA of unknown sequence. Then hybridization pattern is used to obtain DNA sequence. 4. Single nucleotide polymorphism (SNP) and point mutations are detected using DNA chips. DNA chips are used in Human Genome Project for detection of point mutations.
  7. 5. Proteomics DNA chips are useful in this area also. DNA chips are used to identify genes involved in protein-protein interactions. 6. Reverse genetics DNA chips are used in this area where organism complete genome sequence is known. It involves introduction of deletions/insertions or substitutions at will followed by analysis of their fitness. 7. DNA chips are used in genomic miss match scanning (GMS). 8. Drug discovery and agriculture biotechnology are other areas where DNA chips are currently in use.
  8. Future of DNA chips  Major limitation of DNA chips at present is the unavailability of complete genome arrays for higher eukaryotes, including humans.  It is expected that within the next few years such DNA chips will be available.  This will help the biotechnologist to capture the functional snapshot of the genome in action for higher organisms.
  9. Restriction fragment length polymorphism (RFLP)
  10. Restriction fragment length polymorphism (RFLP) • It is another technique based on hybridization principle. • DNA is a polymorphic molecule, i.e.exist in several forms. • DNA of an individual varies from others. Sequence of DNA of an individual is unique. • Further, mutations in DNA generates polymorphic DNA in same individual, which occurs in diseases. • So, DNA polymorphism is due to variations in sequence. • When DNA of an individual is subjected to digestion with restriction enzyme fragments of varying sizes or lengths that are unique to individuals sequence or cell are produced.
  11. • RFLP may also result from presence of variable numbers of tandem repeats (VNTR) in DNA. • These are short sequences of DNA that are scattered locations in genome and repeated in tandem. • The number of these repeats are unique to individual. • When DNA of two individuals is subjected to digestion with restriction enzymes fragments that vary in length and number are generated. • Therefore, RFLP of two individuals results from the differences in the location and number of cleavage sites. • Differences in DNA of two individual may be due to evolutionary changes.
  12. • RFLP is similar to southern blotting in many aspects. • Initial step of RFLP involves digestion of more DNA samples with restriction enzymes where as in southern blotting only one DNA sample is digested. • Rest of the steps of RFLP are those of southern blotting. • Hence, in RFLP next step probes are used for hybridization. • Probes hybridizes with fragment containing complementary sequences. • Then polymorphisms are detected by presence or absence of bands after hybridization.
  13. Methods for detection of RFLPs Two common methods are: 1. Southern hybridization 2. Polymerase chain reaction (PCR)
  14. Applications 1. RFLP is used as a diagnostic test of inherited disease. For example, HbS: In HbS gene there is loss of one restriction site for restriction enzyme due to mutation where as normal HbA gene has two cleavage sites. So, RFLP of sickle-cell anemia patient shows two bands where as in RFLP of normal individual three bands appear.
  15. 2. RFLP is also used to identify chromosomal difference. 3. RFLP is used for isolation and sequencing of closely related genes. 4. RFLP is combination with PCR is used to detect DNA variations.
  16. DNA finger printing
  17. DNA finger printing It is the present day genetic detective in the practice of modern medical forensics. History:- original DNA fingerprinting technique was developed by Alec Jaffreys in 1985. It is commonly used a more general term DNA profiling is preferred.  DNA fingerprint is an analysis of the nitrogenous base sequence in the DNA of an individual.
  18. DNA finger printing • Use of finger prints in crime detection (forensic science) is well known. • Like finger prints, DNA of an individual is characteristic of that individual. • In other words, DNA make up of every human is different. • DNA finger printing is a technique used to identify an individual from DNA obtained from blood or semen or piece of tissue.
  19. • The technique is based on identification of tandem repeats of satellite DNA. • Satellite DNA consist of repetitive sequence. For example, satellite DNA of fruit fly consist of repetitive sequence ACAAA CT. • The length of repetitive sequence or satellite DNA varies and depends on the organism or individual. • In addition, satellite DNA is repeated several times (107) and present on all chromosomes of genome
  20. steps of DNA finger printing 1. DNA of suspect is cleaved with restriction endonuclease, which does not cut satellite DNA. 2. The fragments are separated on agarose gel electrophoresis and southern blotted. 3. Satellite DNA is identified by radiolabelled satellite DNA probe.
  21. 4. Probe hybridizes with DNA fragments containing satellite DNA. 5. The number of satellite DNA present in a given fragment depends on the size of fragment. The size of fragment in turn depends on the individuals DNA. It varies from individual to individual. 6. Southern-blot pattern thus obtained is unique to individuals just like finger print and it is called as DNA finger print. In Figure DNA finger prints of four unrelated people are given. Note that each one has characteristic pattern. With the help of this DNA finger print, sample is matched to individual or suspect.
  22. Applications of DNA fingerprinting  The amount of DNA required for DNA fingerprint is remarkable small.  The minute quantities of DNA from blood strains, body fluids, hair fibre or skin fragments are enough.  PCR is used to amplify this DNA for use in fingerprinting.  It has wide range of applications- most of them related to medical forensics.
  23. Some important are: 1. Identification of criminals, rapists, thieves etc 2. Settlement of paternity disputes. 3. Use in immigration test cases and disputes. NOTE: DNA finger printing and parental dispute • Since half of the child DNA comes from parental DNA, DNA finger prints are used to establish parentage of child by matching childs DNA finger prints with parental DNA finger prints