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DNA SEQUENCING

DNA SEQUENCING . Principles & methods . Topic Outline . Principles of DNA Sequencing . Key Principles of Sequencing. Partial copies of DNA fragments made with DNA polymerase Collection of DNA fragments that terminate with A,C,G or T using ddNTP or chemical. Separate by gel electrophoresis

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DNA SEQUENCING

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  1. DNA SEQUENCING Principles & methods

  2. Topic Outline

  3. Principles of DNA Sequencing

  4. Key Principles of Sequencing • Partial copies of DNA fragments made with DNA polymerase • Collection of DNA fragments that terminate with A,C,G or T using ddNTP or chemical. • Separate by gel electrophoresis • Read DNA sequence

  5. Maxam-Gilbert Method

  6. Manual Method:Dideoxynucleotide Chain Termination

  7. Running the reaction of all the dideoxy nucleotides using different dyes generates this type of diagram in same lane.

  8. Automated Sequencing • Sometimes the spacing between the bands is hard to measure. • Thus use machine to run and read the electrophoresis. • Capillary electrophoresis: the fragments are piped through a tiny glass-fiber capillary during the electrophoresis step, and they come out the far end in size-order.

  9. New method Summary Comparison Old method Chemical cleave method Sequence small fragments of DNA The radioactive labelling is done on the dsDNA. Division of aliquots is done by methylation or removal of base. Requires DNA Breaks DNA at different nucleotides • Enzymatic cleave method • Sequencing small fragments are problematic. • The radioactive labelling is done on the ssDNA. • Allow high throughput automated sequencing techniques. • Allow Real Time detection. • Requires DNA synthesis • Termination of chain elongation

  10. Southern, Northern and Western blotting

  11. Comparison of Southern, Northern, and Western analyses of Gene X

  12. SOUTHERN BLOTTING • The technique was developed by E.M. Southern in 1975. • The Southern blot is used to detect the presence of a particular piece of DNA in a sample. • The DNA detected can be a single gene, or it can be part of a larger piece of DNA such as a viral genome.

  13. Southern hybridization Transfer buffer

  14. Detection of an RFLP by Southern blotting

  15. Flow chart of Southern hybridization Preparing the samples and running the gel Southern transfer Probe preparation Prehybridization Hybridization Post-hybridization washing Signal detection Isotope Non-isotope

  16. SOUTHERN BLOTTING • The key to this method is hybridization. • Hybridization-process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA.

  17. SOUTHERN BLOTTING • There are 2 important features of hybridization: • The reactions are specific-the probes will only bind to targets with a complementary sequence. • The probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.

  18. Comparison of Southern, Northern, and Western blotting techniques

  19. MB206 Mutagenesis

  20. What Is a Mutation? • Genetic information is encoded by the sequence of the nucleotide bases in DNA of the gene. The four nucleotides are: adenine (A), thymine (T), guanine (G), and cytosine (C), a mutation is a change in the order of these nucleotides. • A change in the order can cause the gene to encode for wrong proteins and inhibit the function of the gene or cause the gene to be virtually inactive.

  21. Mutagenesis • Mutagenesis (the creation or formation of a mutation) can be used as a powerful genetic tool. By inducing mutations in specific ways and then observing the phenotype of the organism the function of genes and even individual nucleotides can be determined.

  22. Some types of Mutagenesis • Directed Mutagenesis • Site-directed/Site-specific Mutagenesis • Mismatched Mutagenesis

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