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 formed.

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

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

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

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)

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

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

Using Southern Blots in DNA Fingerprinting

Yesterday’s lab – using PCR for DNA fingerprinting

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

Shotgun Sequencing of a Genome

Gene Chip Technology