The process of hybridisation and how it can be utilised for biological use. PDF

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HYBRIDISATION – how do we identify a particularsequence of DNA/RNA?DENATURATION – separation + coming together of parts of DNA (or RNA).HYBRIDISATION – the complementary DNA sequence is used (doesn’t have to be identical). Annealing of DNA strands.  Use complementary sequence as a PROBE.CHEMICAL D...

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HYBRIDISATION – how do we identify a particular sequence of DNA/RNA? DENATURATION – separation + coming together of parts of DNA (or RNA). HYBRIDISATION – the complementary DNA sequence is used (doesn’t have to be identical).  

Annealing of DNA strands. Use complementary sequence as a PROBE.

CHEMICAL DENATURATION – opposite of hybridisation. It’s the separation of DNA strands (does not break the backbone). THERMAL DEATURATION – heat treatment, denatures DNA + RNA (separated strands). Now DNA is single stranded so has higher absorbance. Follow this experiment with UV absorption:    

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A – T REGIONS: denature first. However, as soon as denature one portion of DNA (the A-T bit), the regions after this point are destabilised. So, once you get to a certain temperature, the whole thing unzips. G-C base pairs more rigid so don’t move around so readily when heat DNA = more resistant to denaturing. Tm is DIRECTLY RELATED to G-C content.  Tm = half-way transition between ds and ss DNA RNA = denature at lower absorbance as not connected in a continuous helix.

UV ABSORPTION = used to measure amount of DNA present (bases absorb light). The max wavelength = 260nm. RENATURATION:   

Need to be slow process in order to be efficient = DNA finds its complementary partner to become ds again If fast = partial hybridisation as can’t find its partner. Done by cooling the DNA OR add agents that will hybridise.

HYBRIDISATION: This needs to occur at a temperature below Tm – mixture of molecules we want to come together just below the Tm. Efficiency of hybridisation (at a given temp) will depend on: a) Length b) G+C content c) How many failures of complimentary pairing = lower temp even more. Target molecules – trying to detect. Probes – shorter known sequence, have fluorescent/radioactive labels. Once bound we can detect presence.

COLONY HYBRIDISATION:  

Linked to the basic cloning methods lecture. Used to detect which colony on the agar plate has a specific sequence.

SOUTHERN BLOT:  Capillary action  Wash a probe of a particular sequence across the DNA to identify which band on the gel contains the sequence. MICROARRAYS/FLUORESCENT IN SITU HYBRIDISATION/IN SITU HYBRIDISATION.

IN VITRO CLONING – PCR: Can be repeated over and over to make lots of copies of DNA fragments OUTSIDE OF A LIVING ORGANISM = NO VECTORS. A mixture of nucleotides, primers, DNA polymerase and DNA samples is set up. 1) Heat to 95 degrees => break hydrogen bonds between bases to produce 2 DNA strands. 2) Cooled to 50 – 65 degrees => allows primers to base pair to their complementary DNA template. 3) Heat back up to 72 degrees => allow DNA polymerase to extend the primer by joining nucleotides together. TEMPERATURE = The temperatures used are dependent on length and CG content of primer. TIME = depends on length of DNA want to make (how far apart opposite primers are). LENGTH = defined by distance between the 2 primers. Forward primers  finishes at 5’ end. Reverse primers  finishes 3’ end. Repeat the process n number of times to produce large quantities of the DNA fragments.  Longer primers are more specific.  Primers encourage DNA polymerase to bind.  DNA polymerase used is from bacteria that live at hot temperatures, so the enzyme doesn’t denature at 72 degrees.  To calculate how many cycles = 2^n.

SEQUENCING How could we interrogate every position? = stopping the polymerisation at every position.

STOPPING THE CHAIN: 

NUCLEOTIDE TRIPHOSPHATE - 3’ attacks the Pi.

(2’,3’) DIDEOXYNUCLEOTIDE (ddCTP) = has a H not OH  replace dATP (OH) with ddATP (H) = can’t proceed any further. Small ddATP = stops at each A = set of DNA fragment of different lengths that all have a A at end = must have a T at this position. HOW DO WE DETECT THE LENGTHS? Radioactive labels on either the: ON PRIMER (ON 5’) or INCORPORATED IN NUCLEOTIDE (better method, label with radioactive Pi = larger chain = more labelling). C + G this time = ddCTP. SEPARATION OF FRAGMENTS VIA ELECTROPHORESIS:   

Denaturing polyacrylamide gel – instead of agarose Containing urea = separate according to length, denatures the DNA Detect radioactive fragments only on X ray film.

SEQUENCING GEL:   

Read from bottom to top. Bottom, shortest fragments – closest to primer. Top, longest fragments – further away.

PI32 = fuzzy results S35 gel = not so active and doesn’t travel as far = clearer results....