HC5 gene cloning and gene manipulation

Question 1

restriction enzyme type 1

Answer 1

cut <1000 bp downstream recognition site

Question 2

restriction enzyme type 2

Answer 2

• mostly used.
• recognises a sequence and cuts within this recognition sites: independent of methylase.
• Recognises palindromic sequences

Question 3

restriction enzyme type 3

Answer 3

One enzyme cuts and methylates a sequence 24-26 bps away. not used for cloning

Question 4

isoschizomers

Answer 4

restriction enzymes recognising the same sequence. they can still cut differently. you can get blunt or sticky ends.

Question 5

DNA ligases

Answer 5

they restory the phosphodiester bond between bases by hydrolysing ATP.

Question 6

cloning of DNA

Answer 6

ligating DNA fragments into plasmid factors –> storage and amplification of specific DNA fragments

Question 7

Cloning vectors have certain specifications: 7

Answer 7

• viral backbone: to facilitate integration of the vector
• selection marker: e.g. antibiotics resistance
• promotor and terminator
• unique restriction/recombination sites
• origin of replication: target gene needs to be amplified
• high copy number: beneficial if plasmid multiplies a lot.
• Reporter system: to establish recombinants, such as GFP.

Question 8

Gateway cloning

Answer 8

use recombination sites instead of restriction sites.

the entry clone: contains DNA fragment of interest flanked by 2 attL (recombination) sequences.

destination vector: contains ccdB flanked bu attR sites.
Add LR clonase –> recombination between attL and attR sites –> get attB and attP sites

DNA is inserted into the vector = expression clone.

This reaction can be reversed by BP clonase.

Question 9

topoisomerase (TOPO) general function + usage in cloning

Answer 9

used to ligate PCR fragments directly into vectors

Topoisomerase relieves the tension on the DNA in front of the replication fork by making a nick in
one or both of the strands so that the DNA in front of the replication fork is not overly coiled

Question 10

cDNA synthesis (eukaryotes)

Answer 10

Start cDNA synthesis by adding a poly T primer (because mRNA has a polyA tail).
- RNA dependent DNA polymerase makes a DNA strand using RNA as a template
- DNA-RNA molecule
-RNA is degraded
- add a second primer
- second strand is made by DNA polymerase = ds DNA