DNA and identification

dna

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difference in DNA

Introns are the regions in the DNA which are used for DNA profiling

within the introns there are short sequences of DNA which are repeated many times to form microsatalites (2 or 3 base pairs smaller)and minisatilites.(20 to 50 base pairs bigger)

the mini or micro satellites appear in the same position on each of the homologus chromosomes. The number of different introns makes huge variation so individual have unique different patterns which can identify them.

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how DNA profiles are produced

A sample of DNA is taken it is chopped up into fragments using an enzyme called restriction endonuclease,

 cuts the DNA at particular points in the intron sequence.

different restriction enzymes cut the DNA at different points of specxific base pairs known as restriction sites.

Using these sites you can cut either side of the mini or micro satellite units leaving the repeated sequence intact

The fragments then need to be seperated and identified which is where gel electrophoresis comes in which is a variation of chromotography.

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Gel electrophoresis

DNA fragments are placed in wells in agarose gel mediumin a buffering solution (to maintain a constant pH) with known DNA fragments to help identification.

the gel contains a dye (EtBr, ethidium bromide) which binds to the DNA fragments in the gel, the dye will then fluoresce when placed under ultra violet light UV allowing easier identification.

this needs a relatively large sample of DNA  and shows up large fragments containing minimum of 50 base pairs a mini satellite.

smaller regions of DNA  and specific genes can now be identified using a technique called sothern blotting and extension to this technique

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Southern blotting

an alkali solution is added to the gel electrophoresis and a nylon filter is placed over it.

dry absorbant paper is used to draw the solution containing the dna fragments from the gel to the filter, leaving the DNA fragments as blots on the filter.

the alkali solution also denatures the DNA fragments so the strands seperate (breaks the hydrogen bonds) the base sequence is then exposed. a single stranded gene probe with a labled radioactive can then be added the probe

 hybridises with the uninque target sequence on the denatured DNA which can then be identified

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a gene probe

a short DNA sequence that are complementary to specific sequences which are being sought.

each probe is labled with either a radioactive element or fluorescent molecule. these are added to the filter in southern blotting binding with the complementary DNA strand exposed due to the aklali denaturing the hydrigen bonds in a process called hybredisation.

each probe can the be washed away and a x-ray can be taken of the filter showing the DNA regions, used to pick out the short tandon repeats which are specific to individuals. 

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(http://cg.scs.carleton.ca/~morin/teaching/compbio/f_s02gelelect.gif)

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Polymerase chain reactions (PCR)

The polymerase chain reaction adopts the natrual process in which DNA is replicated in the cells making it possible to produce enough DNA for a profile from tiny traces of biological matter.

  • DNA polymerase primers must join to the beggining of the sequence of the seperated DNA strand before copying can begin
  • a supply of the 4 nucleoitoid bases are mixed together in the PCR vial and placed in the PCR machine.
  • The mixture is heated to 90-95 degrees which causes the dna strands to seperate (hydrogen bonds break).
  • the mixture is then cooled to 55-60 degrees so the primers bind to the single DNA strands.
  • the mixture is heated to 75 degrees which is the optimum temperature for the DNA polymerase enzyme to build the complementary strands og DNA.
  • this is repeated about 30 times to give billions of copies of the original DNA.
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(http://universe-review.ca/I11-50-PCR.jpg)

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