Top Ten Points I need to know about the Biotechnology Test

1. When transcribing DNA, follow the following steps:

     1. Look for 5’ TATA 3’

     2. Look for AATAA on the coding strand

     3. Look for the intronic sequences

     4. Look for 5’ ATG 3’ on the coding strand

     5. Choose which strand is the coding and template and start transcribing and translating

2. Vector cloning:

For this technique, the gene of interest is isolated from the old organism. The gene and the plasmid is cut by the same restricted enzyme (when orientation is not important). The gene of interest is then attached to the plasmid through DNA ligase. The plasmid is inserted into the bacteria and it is left to grow. Through selection, the bacteria with the desired plasmid are induced into the new organism.

3. PCR (Polymerase Chain Reaction):

The double stranded DNA is mixed with Taq polymerase, primers bind to DNA, and DNA nucleotides. The mixture is heated and cooled several times. During this time, the hydrogen bonds of the double helix is broken and rebuilt, creating more DNA fragments.

4. RFLP (Restriction Fragment Length Polymorphism)

Created by Frederick Sanger, RFLP is a method of DNA sequencing. The DNA in question is mixed with nucleotides, primers, polymerase along with dideoxynucleotides. ddNTPs have no hydroxyl group so they do not react. Four test tubes have the mixture along with the ddNTPs for adenine, guanine, cytosine and thymine. After some time, the four mixtures are run through gel electrophoresis to determine the DNA sequence

5. RFLP always involves COMPLETE digestion by the restriction enzymes.

6. Gel electrophoresis 1

 DNA runs from the top to the bottom as the top is negatively charge and the bottom is positively charged. Since DNA is negatively charged, it is attracted to the positively charged bottom. Since smaller DNA fragments run faster down than large fragments, therefore the bands at the bottom are smaller than the ones up top.

7. Gel electrophoresis 2

A gel electrophoresis consists of:

Ladder, Lane 1, Lane 2, Lane 3, etc.

To complete each lane, one has to know which restriction enzyme is used and whether it is complete or incomplete digestion. If it is complete, all the restriction sites and cut. If it is incomplete digestion, there could be 0 cuts to the number of restriction sites that are on the DNA. Each fragment has a certain about of bp (base pairs) and are labelled as bands on the gel electrophoresis.

8. Restriction enzymes: blunt and sticky

The sticky ends of the gene of interest are needed to place it in the plasmid. Blunt ends however are not designed to be placed on the plasmid. It is used to cut up foreign DNA. An example would be a mosquito putting its DNA into one’s bloodstream. The blunt ends are used to cut them up to prevent disease, infections, etc.

9. Orientation of restriction enzymes

Orientation of the gene of interest is not important when only 1 restriction enzyme is used, as it is cut on both sites of the plasmid. However, orientation is important when 2 restriction enzymes are used to cut one of each side as they both have to match the sites on the plasmid.

10. Objectives

PCR: To amplify a piece of DNA.

Vector cloning: To produce a protein product and to induce a new gene into an organism

RFLP: To determine the DNA sequence.