Gene Cloning Vectors

Question 1

Features of cloning vectors?

Answer 1

1- seq. that will permit the propagation of itself in bacteria or in yeast.
2-a cloning site to to insert the foreign DNA, a site that can be cut by many Restriction enzyme.
3-usually acomplished by selectable markers for drug resistance.

Question 2

Write general steps in cloning..

Answer 2

1-Prepare the vector n the dna to b cloned by digestion with restriction enzyme.
2-ligate (join) the forn dna into the vectr with enzyme DNA ligase.
3-introduce the dna into bacterial cells or yeast by transformation.
4-select cells containing dna by secreening for selectable markers.

Question 3

Name types of cloning vectors?

Answer 3

Plasmid
Phage 
Cosmid
Bac. Artificial chromosome ( BAC ) 
yeast artificial chromosome ( YAC )

Question 4

Define plasmid?

Answer 4

• Small, circular , extrachromosomal dna mol. Found in bacteria
• can replicate of their own.
• madefrom natural plasmids by removing unnecessary segments and adding essential sequences.
• they make excellent cloning vectors

Question 5

Tell some features of pBR322…

Answer 5

• it Is a plasmid,used for ecoli cloning vectors
• first artificial plasmid,1977
• named after mexican creators,p- plasmid,BR- boliver and rodriguez
• 4361 base pair in lenght
• ampR gene: amplicillin res.prt.
• tetR gene:tetracyclin rest.prot.
• 40 restriction sites
• 11 sites lie within tetR gene
• 2 sites for restriction enzyme ( hindIII )(cla I )within promoter of tetR gene
• 6 key restriction sites in ampR gene
• ori encodes 2 rnas ( rna I and rna II )and one protein called ROM or ROP
• the ampicillian resistance gene is a penicillin beta lactamase
• promoters p1 & p2 are for the beta lactamase gene.

Question 6

Define phage?

Answer 6

Phages are derivatives of bacteriophage lamda, a virus which infects ecoli
Linear dna molecules,can replace with foriegn dna
Has high transformation efficiency abt 1000 times greater then plasmid vector
Consist of 8-25 kb

Question 7

The extreme ends of lamda dna is knwn as_____?

Answer 7

COS sites ( 12 nucleotide long )

Question 8

Name assembly proteins of the lamda vector?

Answer 8

Nu1 and A

Question 9

Define cosmids…

Answer 9

Extra chromosomal circular dna
Combines features of plasmid and phages
Cloning limit- 35-50 kb

Question 10

Define BAC.

Answer 10

BACS are based on bacterial mini-F plasmid
Are small pieces of episomal bacterial DNA that give the bacteria to initiate conjugation with adjacent bacteria.
Cloning limit- 75-300 kb

Question 11

Explain YACs..

Answer 11

They are artificial chromosomes that replicate in yeast cells

Question 12

YACs consist of ….

Answer 12

• telomeres TEL
• ori
• a yest centromere CEN
• autonomous replicating sequences ARS
• a selectable marker for identification in yeast cells
• recognition sites for restriction enzyme
• Amp resist gene for selective amplification
• YACs can carry 2 mb of dna frgmnts
• Transformation is very low

Question 13

Write steps YACs cloning?

Answer 13

1- the target dna is partialy digest by ecori and the yac vector is cleaved by ecori and bamhi
2- ligate the cleaved vector segments with a digested dna fragment to form an artificial chromosome
3-transform yeast cells to make a large number of copies

Question 14

Define cloning vector?

Answer 14

A cloning vector is a DNA molecule that carries foreign DNA into a host cell,where it replicates,producing many copies of itself along with foreign DNA.