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10. Beskrive hvordan disse tekniker kan anvendes til genom sekventering. Beskrive formålet med det humane genomprojekt
Devlin, s.184-5, cc.4.4

Sanger sequencing, Southern blotting, restriction enzymes and recombinant DNA are all used in the human genome sequencing.

Stepwise:

1. The entire human DNA is cut by a specific endonuclease.

2. The restriction fragment to be sequenced is cloned into a plasmid vector at a specific site.

3. A primer complementary to the known plasmid sequence is used for replicating the DNA fragment of interest.

4. Besides normal nucleotides, specific dideoxy analogs are added to the mixtures, terminating the newly synthesized strands randomly.

5. The result is DNA-fragments of different lengths, each ending with a specific base – showing the precise location of its complementary base in the template DNA.

6. The restriction fragments can then be separated with gel electrophoresis on basis of their length. The DNA sequence can be then read from the electrophoresis bands.   

The human genome project (HUGO) is a large international effort aimed at determining the sequence of the entire human genome of 3.200.000.000 base pairs. Other aims include:

• the creation of physical and genetic maps of the genome,

• sequencing the genomes of modern organisms,

• developing technology for sequencing and informatics for understanding the sequence

• training of scientists in use of these data

• exploring the legal, ethnical, and social implications of the data 

Along with the completion of the human genome sequence, there is a major effort to find and catalogue polymorphisms. 

Chromosome walking - another method using for gene sequencing:

• defines gene arrangement in long stretches of DNA

• provides knowledge of how genes and their regulatory elements are arranged in chromosome

• gives information of how sets of genes can be co-ordinately regulated

Very often it can happen that a gene is too long to be inserted into a vector. Instead, the gene is cleaved with different restriction enzymes. The cloned fragments will contain overlapping sequences with other cloned fragments.

When the gene sequence is to be read, one cloned fragment is taken, screened and then cleaved with another restriction enzyme. The new restriction fragment is purified and complementary sequence is created– the gene is subcloned. The complementary sequence is then used to find the respective fragment which is also found in the next cloned fragment of the original DNA. This process is called re-screening. These processes are repeated until the entire DNA sequence is retrieved and the process is called chromosome walking.

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