34. Restriction endonucleases have been used to introduce the phenylalanyl tRNA
ID: 46398 • Letter: 3
Question
34. Restriction endonucleases have been used to introduce the phenylalanyl tRNA synthetase gene into the plasmid pREP4 from a company called Qiagen, yielding the plasmid called pRO148. The gene was introduced into the plasmid using the restriction endonuclease PvuII, which cuts at the sequence CAG/CTG, and yields blunt ends: CAG and CTG. When you cut the plasmid with PvuII, however, you achieve two fragments that are almost equal in size, and nearly impossible to separate one from the other on an agarose gel. But, based on the sequence, you find that one fragment can be further digested with BglI, which recognizes the sequence:
GCCNNNNNGGC, yielding the fragments GCCNNNN and NGGC. The plasmid is shown below.
(a) Draw the expected banding pattern one would observe on a 1.0 % agarose gel, after digestion.
(b) From the digestion with BglI, diagram the ends of the DNA fragments produced by BglI digestion.
34. Restriction endonucleases have been used to introduce the phenylalanyl tRNA synthetase gene into the plasmid pREP4 from a company called Qiagen, yielding the plasmid called pRO148. The gene was introduced into the plasmid using the restriction endonuclease PvuII, which cuts at the sequence CAG/CTG, and yields blunt ends: CAG and CTG. When you cut the plasmid with PvuII, however, you achieve two fragments that are almost equal in size, and nearly impossible to separate one from the other on an agarose gel. But, based on the sequence, you find that one fragment can be further digested with BglI, which recognizes the sequence: GCCNNNNNGGC, yielding the fragments GCCNNNN and NGGC. The plasmid is shown below. (a) Draw the expected banding pattern one would observe on a 1.0 % agarose gel, after digestion. (b) From the digestion with BglI, diagram the ends of the DNA fragments produced by BglI digestion.Explanation / Answer
(a)
The restriction endonuclease PvuII digests the DNA at
CAG
|
CTG
GTC
|
GAC
The two fragments obtained after PvuII digestion are equal in size and cannot be separated on an agarose gel. For this reason, one of the bands is digested with BglI, to get three bands of three sizes.
The expected banding pattern one would observe on a 1.0 % agarose gel is based on the size of the bands. Of the three, the one that is smaller is size moves faster and it will be viewed below the other two bands. The one is the larger remains at the top in the gel.
(b)
From the digestion with BglI, the ends of the DNA fragments produced by BglI digestion will be as follows:
GCCN
NNN
|
NGGC
CGGN
|
NNN
NCCG
Enzyme activity is affected by CG methylase depending on the sequence following the recognition site. This reaction is favourable at the temperature 37oC.
Source of this endonuclease is Bacillus globigii.
CAG
|
CTG
GTC
|
GAC
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