Study notes for the Biotech Test

Blog 4: Top Ten Points I Need to Know About the Biotech Test

1) RFLP

Restriction Fragment Length Polymorphism

RFLP is the comparison of lengths of DNA. Restriction enzymes are used to completely digest the strands of DNA. Furthermore, using gel electrophoresis with a thick matrix, the differences

of the lengths of DNA amongst individuals can then be identified.

• involves the comparison between the length DNA fragnments between individuals.

2) PCR

Polymerase Chain Reaction

This is a method of DNA replication. It is used to make numerous copies of a desired gene fragment without placing it into a plasmid.

During this method, heat i used to separate the strands of DNA. DNA is subjected to temperature between 94-96 degrees Celsius, causing the hydrogen bonds to break down and separate. The temperature is then brought down to about 50- 60 degrees Celsius. A 5'-3' DNA primer is then added to anneal with the template DNA. After a Taq Polymerase DNA is added at 72 degrees. It adds DNA nucleotides until it reaches the end of the DNA. The processes is repeated until an exponential amount of DNA has been produced.

Heat -> brek hydrogen bond -> cool -> primers -> heat

• Required during PCR
• Taq polymerase + 2 DNA primers + dNTPs (Deoxynucleoside triphosphate) + PCR machines

3) Sangers method of Sequencing

This was a method to sequences the genome of a DNA.

First the DNA template must be replicated several times. then a Radioactive primer must be placed on to the strands. then placed into four reaction tubes. Each tube containing a full supply of nucleotides in form of all four dNTPs.

Now in each of these 4 tubes, each one should have a low concentration of the same ddTTP.

the dideoxy analogue blocks further growth of the chain. After placing these sequences

through a gel electrophoresis. we are able to sequence them.

4) Vector cloning

• Isolate the DNA from the organism with the desired gene.
• Then the DNA is fragmented with restriction enzymes, generating cuts with sticky ends
• DNA fragments are incorporated in to bacterial plasmids
• The plasmid is cut with the same restriction enzyme that generates the same cohesive ends
• the cohesive ends of the gene and the cut at on the plasmid are joined together by DNA ligase used to form covalent bonds
• place the plasmid back into the bacterial host.
• the cells can now be grown or placed in the organism of desire