Chapter 8: ICSD

8.1 DNA Cloning
Processes:

Obtaining cDNA library:
1. Total mRNA is isolated from selected tissue using column chromatography.
2. mRNA are reverse transcribed into cDNA using reverse transcriptase.
3. Recombinant DNA are transformed into bacterial cells then grown on plates of nutrient agar.

Reverse Transcription:
To form cDNA library.
1. Oligo(dT) sequences act as primers bind to mRNA poly(A) tail for reverse transcription.
2. Reverse transcriptase synthesises a single DNA strand by adding deoxyribonucleotides to free 3' OH end of the primer, using mRNA as a template, forming RNA-DNA duplex.
3(a). Certain reverse transcriptase synthesises a hairpin loop at 3' end of first cDNA strand.
4. Ribonuclease H is added to degrade mRNA template of RNA-DNA duplex.
5. DNA polymerase is added to synthesis second cDNA strand, using hairpin loop as primer, first cDNA as template.
6. S1 nuclease is added to cut the hairpin loop to form ds cDNA.
3(b). Ribonuclease H is added to degrade mRNA template of RNA-DNA duplex.
4. Terminal transferase is added to add multiple cytosine residues to 3' end of first cDNA strand.
5. Oligo(dG) acts as primer for synthesis for second DNA strand.

Screening of Gene Library: via Molecular Hybridisation using a Radioactive Gene Probe
To find out which bacteria colony has GoI.
1. Nitrocellulose membrane is laid on master plate. Some bacteria from each colony adheres with membrane.
2. Bacteria cells on membrane is lysed using detergent.
3. dsDNA is denatured into ssDNA using dilute NaOH. Denatured ssDNA adheres to membrane.
4. Membrane is baked at 80 degrees celsius to fix ssDNA onto membrane.
5. Membrane is submerged in radioactive probe solution, hybridising it with GoI via comp bp forming H bonds.
6. Membrane is washed to remove excess unbound probes.
7. Membrane is exposed to photographic film. Colonies with hybridised probes will show us as dark spot on the autoradiograph.
8. Location of bacteria colonies with GoI is identified by comparing with master plate.

Isolation of GoI using RE:
To cleave out GoI before ligating it into new vector for efficient large scale production.
1(a). RE is added to make asymmetrical cleave of sugar phosphate backbone of both strands of dsDNA to produce sticky ends.
1(b). RE is added to make symmetrical cleave of sugar phosphate backbone of both strands of dsDNA to produce blunt ends.
(i) Linkers are ligated to blunt ends using DNA ligase, then cleaved using RE to produce sticky ends.
(ii) Poly(dN) tail is added to 3' end of GoI using terminal transferase to form sticky ends.
2. Vector is cut using the same RE, producing comp sticky ends. GoI and vector can anneal by forming H bonds btw comp bp.
3. DNA ligase is added catalyse formation of phosphodiester bonds to seal sugar phosphate backbone, forming recombinant plasmid.

Transformation:
1. Bacterial cells are induced to be competent to increase transformation efficiency.
2. Add to ice bath, cold CaCl2 mixture of E.coli and recombinant DNA.
3. Heat shock in 42 degrees celsius water bath for 1-2 min to create temporary poration in cell membrane, allowing plasmids to enter.
4. Mixture is returned to ice bath immediately to close pores, preventing plasmids from leaving.

Selecting Transformed Recombinant Host Cells:
Blue/white Colony Screening (X-gal blue>white):
1. Untransformed bacteria did not take up plasmids with ampR gene will be killed by ampicillin and unable to grow in nutrient medium.
2. Recombinant bacteria will have lacZ gene disrupted and inactivated as insertion of GoI causes insertional inactivation of lacZ gene.
3. Recombinant bacteria cannot produce functional B-galactosidase and cannot convert X-gal into blue compound, remaining white.
4. Non-recombinant bacteria will have intact and active lacZ gene, can convert X-gal into blue compound, appearing blue.

Replica Plating (Tetracycline):
1. Untransformed bacteria cells did not take up plasmids with ampR gene will be killed by ampicillin and unable to grow in nutrient medium.
2. Sterile velvet cloth transfers some bacteria cells from master plate to replica plate containing tetracycline.
3. Recombinant bacteria has tetR gene disrupted due to insertional inactivation will not grow on replica plate.
4. Non-recombinant bacteria has intact tetR gene and able to grow on replica plate.

8.2 DNA Analysis and Genomics
Processes:

Polymerase Chain Reaction (PCR)
To replicate short DNA sequences.
1. DNA Denaturation:
Target DNA and reagents heated to 95 degrees celsius, increasing kinetic energy resulting in breaking of H bonds btw comp bp, to denature and separate dsDNA into ssDNA.
2. Annealing:
Mixture cooled to 55 degrees celsius in excess primers, binding forward and reverse primers to comp sequences flanking target DNA via H bonding btw comp bp.
3. Elongation:
Temperature increased to 70 degrees celsius, Taq pol synthesises comp DNA strand by adding free deoxyribonucleotides to free 3'OH end of primers using target DNA seq as template.
4. Repeat 20-30 times.

Gel Electrophoresis (Agarose/ Polyacrylamide)
To separate DNA based on molecular size across gel at different migration rates.
1. Agarose powder is dissolved in TBE buffer.
2. Agarose is cooled and hardened to form agarose gel of 0.8%-2%.
3. Gel is placed in gel chamber and submerged in TBE buffer.
4. Comb is removed, forming wells.
5. DNA molecules are mixed with loading dye and tracking dye (methlyene blue/ ethidium bromide), loaded in wells of agarose gel near negative electrode.
6. Voltage between 90V-150V is applied across the gel. The buffer conducts a direct current across gel.
7. DNA fragments are -vely charged due to phosphate groups and migrate across gel from negative electrode to positive electrode.
8. Agarose gel acts as molecular sieve to separate DNA molecules by molecular size. Small fragments are less impeded by the gel and migrate faster than large molecules.
9. After electrophoresis, DNA fragments of same molecular size form a band in the gel.
10. DNA ladder is compared with to indicate size of separated DNA.

Southern Blotting
To locate target DNA fragment after gel electrophoresis.
1. Nitrocellulose membrane is laid over electrophoresised gel containing seprated DNA fragments.
2. Gel is supported on layer of sponge in alkaline buffer solution.
3. Paper towels are stacked on nitrocellulose membrane to wick buffer through the gel and nitrocellulose paper by capillary action.
4. As buffer passes through gel, it denatures dsDNA to ssDNA and adheres ssDNA from gel to nitrocellulose paper.
5. Nitrocellulose paper placed in sealed bag of radioactive probe solution with base sequence comp to target DNA sequence.
6. Mixture is incubated to allow radioactive probes to hybridise with target DNA seq via comp bp w H bonds.
7. Excess unbound probes are washed away.
8. Nitrocellulose membrane is autoradiographed by exposing to photographic film. DNA band which is hybridised shows up as dark bands on autoradiograph.