Embed Size (px)
DESCRIPTION
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 - PowerPoint PPT Presentation
Citation preview
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
