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

6. 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

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

8. 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)

9. 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

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

12. Using Southern Blots in DNA Fingerprinting

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

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

15. Shotgun Sequencing of a Genome

16. Gene Chip Technology