Lecture 18

DNA Technology

1. Cut DNA with restrictionenzyme(s).

2. Combine fragments in atube with ligase.

3. Recombinant DNA moleculeformed.

Restrcition Enzymes:-cut specific sequences of DNA-generally 6-bp sequences-generally palindromic-can create blunt ends or

sticky ends

inserting DNA sequencehere disrupts the LacZgene – cannot makefunctional enzyme

lacZ gene: codes for -galactosidase enzyme -galactosidase cleaves X-gal to produce a blue coloured product

mRNA

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 molecule4. 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.

cDNA

RT

PCR

amplified sequenceof interest

A

A

ligation

TA palsmid: a linearized plasmid with free T nucleotides that can stick to thefree A nucleotides on the amplified sequence

inserting DNA sequencehere disrupts the LacZgene – cannot makefunctional 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 PCRfor DNA fingerprinting

Dideoxy Chain Termination Method:DNA Sequencing

O

HOH

O

HH

deoxyribose

dideoxyribose

Shotgun Sequencing of a Genome

Gene Chip Technology