Lecture 18
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Transcript of Lecture 18
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Lecture 18
DNA Technology
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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
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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
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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
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inserting DNA sequencehere disrupts the LacZgene – cannot makefunctional enzyme
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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)
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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
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Southern Blotting – looking for particular sequences of DNA in a mixture
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Using Southern Blots in DNA Fingerprinting
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Yesterday’s lab – using PCRfor DNA fingerprinting
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Dideoxy Chain Termination Method:DNA Sequencing
O
HOH
O
HH
deoxyribose
dideoxyribose
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Shotgun Sequencing of a Genome
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Gene Chip Technology