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Biotechnological tools used for diagnostic
- 1. Biotechnological tools used fordiagnosticS Dr. Sunita Dr. Mrinalini Saran
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- 14. PCR:- A processused to artificially multiply a chosen piece of genetic material. May also be known as DNA amplification. One stand of DNA may yield 230 strands or more.
- 16. Uses of PCR- DNA sequencing Gene cloning DNA profiling Transformation Making artificial genes
- 17. DNA selection DNAis selected either as a complete chromosome or a fragment. Primers are constructed that will bind within a desired region(purple). Additional reaction chemicals are added.
- 18. The reaction mixture Water and pH buffer. DNA to be multiplied RNA Primers Nucleotides DNA Polymerase (Taq)
- 20. Splitting DNA DNAis heated to 950c which causes double stranded DNA to become single stranded.
- 21. Adding Primers RNAprimers are prepared that base pair with a selected sequence of DNA. Two primers must be used. One for each strand of DNA. The reaction temperature is lowered to 600c to allow the primer to attach(anneal).
- 22. Adding nucleotides Thereaction temperature is raised to 720c to allow nucleotides to be added. Polymerase enzyme (Taq) catalyze the addition of nucleotides. Nucleotides are added in a 5´ to 3´ direction.
- 23. Repeating the cycle In the next cycle the heating and cooling steps are repeated. The original (red/purple) strands reproduce as per the first cycle. The new strands only duplicated between the primer site to produce blocks of a set length.
- 25. Continuing the cycle The cycle is then repeated over and over again. With each cycle the no. of short fragments rapidly increases while the no. of larger fragments increases slowly.
- 26. 30 cycles After30 cycles, if replication has occurred fully, a total of 2,147,483,648 strands could be produced. All but 62 will be the short length of the desired DNA fragment.
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- 40. DNA SEQENCING Determiningthe order of bases in a section of DNA. To analyze gene structure and its relation to gene expression as well as protein conformation.
- 41. Purpose Deciphering “code of life”. Detecting mutation. Typing microorganisms. Identifying human halotypes . Designating polymorphisms.
- 42. Maxam and Gilbertmethod A.M.Maxam and W.Gilbert-1977 The sequencing of a double stranded or single strand DNA molecule is determine by treatment with chemicals that cut the molecule at specific nucleotide position.
- 43. Sanger method termedas chain termination method This method uses dideoxynucleotide triphosphates (dint's) chain termination : Which have an H on the 3’ carbon of the ribose sugar instead of the normal OH found in deoxynucleotide triphosphates(dNTPs). There for in a synthesis reaction , if a dideoxynucleotide is added instead of the normal deoxynucleotide , the synthesis stops at that point because the 3’OH necessary for the addition of the next nucleotide is absent.
- 44. comparison 1 Sanger method Enzymatic Requires DNA synthesis Termination of chain elongation Automation Single-stranded DNA 2 Maxam Gilbert Method Chemical Requires DNA Break DNA at different nucleotides Automation is not available Double-stranded or single- stranded DNA
- 45. Applications of DNAsequencing Forensics: to help identify individual has a different genetic sequence. Medicine: can be used to help to detect the genes which are linked to various genetic disorders such as muscular dystrophy. Agricultural : the mapping and sequencing of a genome of microorganisms has helped to make them useful for crops and food plants.
- 46. Advantage:- Improve thehealth care. Helping plants and animals to be able to resist certain disease. Helps in forensic science for identifying criminals.
- 47. Disadvantage:- The riskthat the DNA sequencing may not work right and provide the wrong DNA sequencing of the fragment.
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- 50. DNA Cloning Techniques Technique for gene cloning enable scientists to prepare multiple copies of DNA pieces. DNA pieces are stored in DNA libraries for easy identification and accessibility. DNA cloning can occur (in vitro) via RECOMBINANT DNA TECHNOLOGY and POLYMERASE CHAIN REACTION (PCR).
- 51. MATERIALS AND METHODS Reagents-1) Taq enzyme 2)10X buffer Teq w/o Mgcl₂ Restriction enzyme -1) Xbal and Hind III 2)T4 DNA ligase Scarlett Barley Malt was provided by Zhong liang Malt Division Wheat Malt obtained from Weyermann .
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