manipulating genomes

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Polymerase chain reaction (PCR)

is a version of the natural process by which DNA is replicated

the DNA sample to be amplified, an excess of the four nucleotide base A T C G

the temperature with in the PCR machine is carefully controlled and rapidly at programmed intervals

the reaction can be repeated many time by the PCR machine

30 repeats gives around 1 billion copies of the original DNA sample

makes copies that could then be used in pyrosequecing

Separating the strand: PCR machine increase temperature to 90-95 degrees for 30 second. This denatures the hydrogen between the DNA nucleotide bases so they get separated

Annealing of the primers: temperature is decreased to 55-60 degrees sot he primers can bind to the end of the DNA strand. they are needed for replication of the stand occur

synthesis of DNA: temperature is increased to 72-75 degrees for 1 mins. this is the optimum temperature for DNA polymerase to work. It adds the complimentary bases. this produces an identical DNA strand. The enzyme Taq polymerase is used, which is obtained from thermophilic bacteria found in hot springs

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DNA sequencing: a technique that allows genes to be isolated and read

principles of DNA sequencing

the process involves the terminator bases, modified versions of the 4 nucleotide bases A, T, C, G

this will stop DNA synthesis what they are included

the terminator bases are also given coloured fluorescent tags

A- green

G- yellow

T- red

C-blue

DNA for sequencing is mixed with a primer (DNA polymerase) and an excess of normal nucleotides

mixture placed in thermal cycler - PCR and there is a rapid change in temperature and the cycle is repeated

60 degrees the DNA polymerase starts to build up new DNA strands by adding nucleotide with complementary bases to the single strand DNA template

when terminator bases are added no more bases are added. many different lengths of fragments get produced that are then separated due to their length. the different colours are picked up by lasers thus the order of sequences

the order o bases in the capillary tubes show the sequences of the new, complementary strand of DNA. this can then be used to find the original sequence.

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pyrosequencing

A long length of DNA to be sequenced is mechanically cut

These lengths are then degraded into single strands of DNA and immobilised

A sequencing primer is added and the DNA is incubated with enzyme DNA polymerase, ATP sulfurylase, luciferase and apyrase and a nucleotide-triphosphate/ activated nucleotide (T-TP, A-TP, C-TP, G-TP). When the nucleotide is added a light is generated the more added the more light is given out. (you add each of nucleotide-triphosphate at a different time)

(a) once a activated nucleotide is incorporated into a complementary strand of DNA using the strand to be sequenced as a template. (b) the 2 extra phosphates are released (c) ATP sulfurylase will only work in the presences of APS and coverts the 2 phosphates to ATP (d) in the presence of the ATP the enzyme luciferase converts luciferin to oxyluciferin (e) this creates a visual light that is detected

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separation of nucleic acid fragments by electrophoresis

DNA fragments are put into well the agarose gel strips

this will also contain a buffering solution to maintain a constant pH

in one or more wells (1st or last) are DNA fragments of known length are used

an electric current is passed through the electrophoresis plate the DNA fragments move from the cathode to the anode end

this is due the negatively charged phosphate groups in the DNA fragments

the rate movement depends on mass and length

over a period of time the smaller fragments travel further

the gel gets placed in a alkaline buffer solution to denature the DNA fragment

the two DNA strands of each fragment separate

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The uses of DNA profiling

uses int he filed of forensic science for criminal investigations

PCR and DNA profiling is performed on traces of DNA left at crime scene

it is then compared to suspects DNA

the paternity of child

increasingly used to demonstrate evolutionary relationship between 2 species