Question

 

A) You would like to sequence the genome of this organism. The restriction enzyme cut site for BglII is AGATCT. You will use it to clone fragments of the genome in order to sequence it. How many times would you expect this enzyme to cut within the chromosome of Andrebacillus wieczorius ? (2)

B) You decide to use a modified bacteriophage vector that can accommodate fragments digested with BglII. You use a partial digest on the chromosomal DNA. What are two reasons discussed in class that you need to use a partial digest in order to achieve your overall goal? (2)

C) Astonishingly, this is the first bacterium identified to generate a polyA tail on its transcripts! Wow! Considering that only 4% of the organisms’ genome codes for proteins, briefly describe how you would build a library of only the fractions of the genome that are rich in ORFs. In your answer, describe what type of a library this is called, the name of the technique,

Answer 1

A) The restriction enzyme, BglII has the restriction site of AGATCT where it cuts between A and G nucleotide phosphodiester bonds. The number of times this site is expected to cut within the chromosome of the organism will be dependent on the number of times this site is present in the chromosomes. And also not all the sites will be cut accurately by this enzyme as the DNA modifications will prevent it from cutting the chromosome sites accurately. Hence the number of times the enzyme will likely cut this DNA will be equal to the number of times this site is repeated in the chromosomal DNA.

B) Partial digestion is used in cloning. When you need to clone a DNA fragment that contains a specific restriction site also elsewhere than in the desired position (e.g. you need to use this enzyme to cut at the end of the fragment but not inside), you do partial digestion (you use a lower amount of the enzyme) and generate a mixture of fragments that have stochastically cleaved at these sites, just one site per DNA fragment. A subpopulation of this mixture contains the right DNA fragments with the internal restriction site intact and the site close to the end cleaved, and you use this fraction for cloning (for the other end of the fragment another enzyme is used, so you can do directional cloning). Hence the two reasons can be summarised as: partial digest generates stochastically cleaved sites which help in cloning and also they help to know the location of the restriction sites.

C)If a polyA tail is present then to clone these 4% of organisms genome that codes for proteins, we use a poly dT containing primers that automatically amplify these 4% DNA sequences and also help in generation of the clones. Such a library of clones generated by using poly dT primers is called as cDNA library as only coding regions of the DNA will be cloned.