Lecture 5+6 - Cloning a Gene/Gene Identification

Question 1

What is PCR?

Answer 1

Polymerase chain reaction is the amplification of DNA sequences in the lab. PCR requires two primers complimentary to each strand of DNA and a DNA polymerase.

Repetitive heating (denaturing) and cooling (annealing) and DNA synthesis (extension) amplify the DNA between the primer binding sites which yields larger quantities of DNA.

Question 2

What species can be used as cloning vectors?

Answer 2

Plasmids, Phages (M13, λ), Phagemids, Cosmids, Artificial chromosomes (YACs, PACs, BACs, HACs)

Question 3

What are YACs?

Answer 3

YAC = Yeast artificial chromosomes

A special linear cloning vector in the form of a yeast artificial chromosome, constructed using chromosomal components including telomeres (from a ciliate), and centromeres, origin of replication, and marker genes from yeast. YACs are used to clone long stretches of eukaryotic DNA.

Question 4

What is the role of the telomeres in YACs?

Answer 4

The telomeres stabilise the chromosome ends.

Question 5

How large can the inserts be that are put into YACs?

Answer 5

Very large between 100kb and 10Mb.

Question 6

What advantages do YACs provide over plasmids as cloning vectors?

Answer 6

Plasmids are relatively small and therefore easy to separate from the host bacterial chromosome and they have relatively few restriction sites. However plasmids can only use bacteria as a host.

YACs (yeast artificial chromosomes) contain telomeres, an origin of replication, and a centromere and are extensively used to clone DNA in yeast. With selectable markers (TRP1 and URA3) and a cluster of restriction sites, DNA inserts ranging from 100 kb to 1000 kb can be cloned and inserted into yeast.

Since yeast, being a eukaryote, undergoes many of the typical RNA and protein processing steps of other, more complex eukaryotes, the advantages are numerous when working with eukaryotic genes.

Question 7

Plasmids and cosmids suffer from what limitation?

Answer 7

They can only use bacteria as hosts.

Question 8

What do YACs contain?

Answer 8

A yeast autonomously replicating sequence (ARS)
A yeast centromere (CEN4)
A yeast telomere (TEL)
Genes for YAC selection in yeast (URA3, TRP1)
A supressor tRNA gene (SUP4)
Bacterial replication origin and selectable marker gene

Question 9

When YACs are cut with EcoRI and BamHI what happens to SUP4?

Answer 9

SUP4 is destroyed and two fragments are created

Question 10

When SUP4 expressed what colour colonies are produced?

Answer 10

The expression of SUP4 in ade2-ochre mutant yeast suppresses the mutation and results in the formation of WHITE colonies.

Question 11

PACs are based on what?

Answer 11

PACs are based on P1 bacteriophage.

Question 12

How many origins of replication do PACs have?

Answer 12

They have two replication origins.
One to control lytic DNA replication (P1 lytic replicon)
One to replicate as a plasmid (P1 plasmid replicon)

Question 13

In PACs what is pac?

Answer 13

pac is a site cleaved prior to insertion of phage DNA into phage particles.

Question 14

In PACs what is sacB?

Answer 14

sacB encodes for levansucrase that is toxic for E. coli cells growing on sucrose.

Question 15

How much DNA can PACs hold?

Answer 15

PACs can hold up to 150kb of DNA

Question 16

What are PACs cut with?

Answer 16

BamHI and ScaI

Question 17

How is DNA inserted into PAC vector fragments?

Answer 17

Large genomic DNA is ligated into the BamHI site. The DNA is then packaged in vitro.
Then transfect Cre+ E. coli cells

Question 18

What are BACs?

Answer 18

BACs are bacterial artificial chromosomes, they are engineered version of F’ plasmids

Question 19

What are the problems arising from the use of BACs?

Answer 19

They produce a low DNA yield.

Question 20

What are DNA libraries?

Answer 20

DNA libraries are pools of recombinant DNA molecules. They contain fragments of all DNA sequences present in the genome

Question 21

What are cDNA libraries?

Answer 21

cDNA libraries contain DNA copies of mRNA and are tissue and developmental stage specific. Their formation is dependent on an RNA-dependent DNA polymerase, reverse transcriptase (RT)

Question 22

List the different cloning vectors by size of DNA insert, largest -> smallest.

Answer 22

YAC (0.2-2Mb)
BAC
PAC
Bacteriophage P1
Cosmid vectors 
Bacteriophage lambda replacement vectors
Bacteriophage lambda insertion vectors 
Standard plasmid vector (0-10kb)

Question 23

How is a genomic library constructed?

Answer 23

A high molecular weight piece of DNA (>100kb) is is partially restriction digested to a size of 20kb. (partial digestion produces sticky ends which results in more efficient ligation than when the ends are blunt.

The DNA fragments are then treated with DNA methyl’s to protect them form restriction enzyme cleaver treatments with EcoRI methylase.

Oligonucleotide linkers are then added to the methylated DNA in large excess in the presence of high concentration of DNA ligase.

The genomic DNA is then digested with restriction enzymes, for example Sau3AI which generates DNA fragments that are compatible with the sticky end produced by BamHI cleavage of a vector.

Once the DNA fragments are produced they are cloned into a suitable vector.

The recombinant vector and insert combinations are then grown in E. coli. E. coli cells infected with either a lambda phafe or transformed with a plasmid DNA are unable to support the replication of additional DNA molecules of the same type. Consequently, each lambda plaque or bacterial colony contains multiple copies of the same recombinant DNA molecule.

A library of these molecules is produced by pooling colonies or plaques such that sufficient are present to ensure that each genomic DNA fragment is represented at least once within the library. Using large DNA fragments means less fragments must be formed.