Answers to Gene technology exam 2011-10-18

1)

a)  Competent cells: Cells treated to be permeable for uptake of DNA

b)  Transfection: Introduction of DNA into bacteria using non-viral metods also called transformation

c)  Phagemid: Combination of phage and plasmid containing F1 origin to be able to obtain SS-DNA

d)  Operons: Regulation sequence containing promoter-operator--structure genes and terminator signal

e)  Cos-sites: Sequence that give single stranded base overhang, the size of the DNA between the cos sites determines if it can be packed into phage particles.

2)

a)  Use Tetracyclin plate for transformation and do a replica-plating on an Agar-Amp plate- Colonies that are not visible on agar-Amp plate (but on the tetracycline plate) will have the insert.

b)  Religation of the vector without any insert or that the restriction enzyme did not work.

c)  For expression of genes the gene can be inserted in wrong direction, also the vector can more easily be relegated.

d)  Alkaline phosphatase removes phosphate group from 5’-end. This improves the ligation frequency by hindering a possible religation of the vector.

3)

a)  cDNA originates from mRNA i.e the expression of mRNA in a certain tissue. Genomic library contain all DNA, including introns, cut into pieces and cloned into a vector.

b)  Look at fig 8.7 page 135 in Gene Cloning book (6th edition)

c)  Design primer based on known DNA sequence from DNA database or based on conserved sequences from related species.

4)

a)

Forward primer:

5’- ATGACTAACTCCAAGGGTTAC-3’ Tm value 60 C , 21 bases

Reverse primer:

5’- TTAAGCAATAAATTCATAGGGAAC-3’ Tm value 62 C , 24 bases

b) TA cloning need a polymerase that create A base overhang (Taq polymerase)

Vector blunt end cleaved and treated with terminal deoxy nucleotidyl transferase

See also fig 9.12 ipage 156 in Gene Cloning book (6th edition)

c) Transformation control: Used to see if the competent cells are OK use a plasmid with antibiotic resistance and known [DNA].

Ligation control: cleaved vector with no insert and added ligase, to chech the relegation frequency of the vector.

d)

Forward:

5’- ATGACTAACTCCAAGGGTTGGAGACGTGCGACGAGG-3’

Reverse:

5’- CCTCGAGCCACGTCTCCAACCCTTGGAGTTAGT-3’

e) See labmanual site-drected mutagenesis