H2 Biology 11 - Molecular techniques

selectively amplify a specific target DNA sequence

1. mark out section of DNA to be amplified by attaching to complementary bases on 3' ends of target DNA sequence

2. provide free 3' -OH end for Taq pol to ad dNTPs, elongating primer into new strand of DNA

1. r&b to 3' -OH end of primer & adds free dNTPs to primer

2. free dNTPs cbp via H bonds w/ exposed bases in template DNA

3. catalyses formation of phosphodiester bond b/w adjacent dNTPs, synthesising new DNA strands in 5'->3' direction

1. template DNA

2. primers

3. dNTPs

4. Taq polymerase

DAP

Denaturation

heated to 95^oC >

H bonds b/w complementary bases of dsDNA fragments break >

dsDNA separates > ssDNA

Annealing DNA primers

cooled to 65^oC >

primers cbp w/ comp. seqs @ 3' end of target seqs in ssDNA >

primers provide free 3' -OH end for Taq pol to add dNTPs to elongate new DNA strand >

DNA primers added in excess to prevent ssDNA from reannealing

Primer extension

temp increased to 72^oC >

optimum temp of Taq pol. Taq pol synthesises rest of new DNA strand by adding dNTPs to 3' -OH ends of both primers by catalysing formation of phosphodiester bonds b/w adjacent dNTPs >

each primer lengthened into new DNA strand, each newly synthesised DNA strand complementary to target DNA seq, each ssDNA obtained after Denaturation now dsDNA >

no. of dsDNA molecules after n cycles = 2ⁿ

1. highly sensitive; can amplify seq from small amounts of target DNA

2. speed; millions of copies of DNA obtained in short amount of time

3. specificity; amplifies only target seq as primers are specific

4. automation; Taq pol thermostable & doesn't denature @ high temps; fully automated

5. robust; can amplify specific DNA seq from badly degraded DNA

6. easy to use; easy to set up thermal cycler for PCR

1. too sensitive; small amount of contaminant DNA containing seq complementary to primers can be amplified leading to misleading result

2. requires prior info of target DNA seq; info required to design specific primers for selective amplification

3. limit of size of fragment to be amplified; increase in length of target seq decreases efficiency of amplification

4. infidelity of DNA replication; Taq pol no proofreading fn, errors early in PCR get intensified. final product is mixture of similar but not identical target DNA seq

DNA -vely charged due to phosphate groups of sugar-phosphate backbone>

when placed in electric field, -vely charged DNA molecules move to anode away from cathode >

DNA fragments of diff molecular sizes in given sample separated based on diff rate of migration through gel >

larger DNA fragments moves slower through pores, moves shorter distance & found nearer well >

rate of migration inversely proportional to length of DNA fragments (5m)

1. contains -vely charged ions that conduct electricity, allows DNA to move through gel towards anode when current turned on

2. helps maintain relatively constant pH level

CLRV

Casting agarose gel

molten agarose poured into casting tray, solidifies >

wells formed @ 1 end of gel to load DNA samples >

gel soldifies, placed in buffering soln

Loading samples

samples mixed w/ loading dye >

glycerol increases DNA density, sinks to bottom of well >

coloured dye enables locating invisible DNA samples >

loading dye moves to anode, allows for visual tracking of DNA sample during GE >

DNA ladder in 1 well, samples loaded into wells @ cathode >

Running of gel

current turned on, -ve DNA fragments migrate out of well, travel to anode >

DNA fragments of diff sizes separated based on rate of migration

Visualisation

current turned off b4 dye reaches end of gel >

gel stained w/ EB, placed under UV light; EB fluoresces & bands visible

DNA sample cut by restriction enzymes; dsDNA fragments separated by GE >

sponge soaked in alkali soln > gel > nitrocellulose membrane sheet > stack of paper towels >

paper towels draw alkaline soln upwards through gel via capillary action, alkali soln denatures dsDNA by breaking H bonds b/w 2 DNA strands >

ssDNA fragments transferred to nitrocellulose membrane, adhere firmly in exactly same position as in gel

isolate & identify DNA fragment of interest by transferring DNA from gel onto nitrocellulose membrane before carrying out NAH if there are too many bands to distinguish individually

nitrocellulose membrane removed from gel & placed in sealed plastic bag containing RLssDNA probes >

probes complementary to DNA seq of interest, binds via CBP w/ targeted ssDNA fragments of interest on nitrocellulose membrane >

nitrocellulose membrane removed from bag, washed thoroughly to remove unhybridised probes >

autoradiography; X-ray film placed over nitrocellulose membrane >

radioactivity in bound DNA probes exposes film, forms image corresponding to bands hybridised w/ probe