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Lecture 18

Lecture 18. DNA Technology. Restrcition Enzymes: -cut specific sequences of DNA -generally 6-bp sequences -generally palindromic -can create blunt ends or sticky ends. Cut DNA with restriction enzyme(s). 2. Combine fragments in a tube with ligase. 3. Recombinant DNA molecule

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Lecture 18

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  1. Lecture 18 DNA Technology

  2. Restrcition Enzymes: -cut specific sequences of DNA -generally 6-bp sequences -generally palindromic -can create blunt ends or sticky ends • Cut DNA with restriction • enzyme(s). • 2. Combine fragments in a • tube with ligase. • 3. Recombinant DNA molecule • formed.

  3. inserting DNA sequence here disrupts the LacZ gene – cannot make functional enzyme lacZ gene: codes for b-galactosidase enzyme b-galactosidase cleaves X-gal to produce a blue coloured product

  4. 1. Isolate RNA from cells or tissue. 2. Use reverse transcriptase (RT) to synthesize cDNA from RNA. 3. Amplify your gene of interest using PCR – Taq polymerase leaves an additonal A nucleotide at the 3’-end of each amplified DNA molecule 4. Combine amplified DNA with TA-plasmid and ligase. 5. Transform bacteria. Look for white colonies that grow on LB plates with ampicillin and X-gal. mRNA RT cDNA PCR A amplified sequence of interest A ligation TA palsmid: a linearized plasmid with free T nucleotides that can stick to thefree A nucleotides on the amplified sequence

  5. inserting DNA sequence here disrupts the LacZ gene – cannot make functional enzyme

  6. Polymerase Chain Reaction: Amplifying DNA A repeat of 3 basic steps: 1. Denaturation 2. Annealing 3. Extension Result: Exponential increase in the amount of DNA present Need: template primers buffer and salts dNTPs enzyme – Taq polymerase (heat-stable)

  7. DNA Gel Electrophoresis: gel is normally made from agarose like a “sieve” the concentration of agarose determines the size of the “holes” higher concentration = smaller holes lower concentration = larger holes smaller DNA molecules run faster through the gel larger DNA molecules migrate slower an electric field is applied to the gel DNA is negatively charged DNA migrates to the anode

  8. Southern Blotting – looking for particular sequences of DNA in a mixture

  9. Using Southern Blots in DNA Fingerprinting

  10. Yesterday’s lab – using PCR for DNA fingerprinting

  11. Dideoxy Chain Termination Method: DNA Sequencing O H OH deoxyribose O H H dideoxyribose

  12. Shotgun Sequencing of a Genome

  13. Gene Chip Technology

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