Genomic and cDNA cloning

Question 1

What are cosmids?

Answer 1

A hybrid between lambda and a plasmid.

Question 2

What are the benefits of using a cosmid?

Answer 2

It enables large pieces of DNA to be cloned (35kb).

Question 3

How does the cosmid behave and function?

Answer 3

It uses the efficient infection of E.coli cells by lambda particles and once inside the E.coli behaves as a plasmid.

Question 4

What is special about the lambda DNA part of this molecule?

Answer 4

Cohesive ends/cos sites that are 12 base complementary single stranded ends.

Question 5

What is special about the cos sites?

Answer 5

The enzymes that package lambda DNA into the protein coat only require cos sites in order to function, and will package not only lambda DNA but any DNA that carries cos sites separated by 37-52kb of DNA.

Question 6

What is an example of a cosmid?

Answer 6

pJB8. It is 5.4kb in size and has an ampicillin resistance gene, an ori site, a suitable cloning site and a small piece of lambda DNA containing the cos site.

Question 7

What is the benefit of this cosmid?

Answer 7

Allow us to clone large pieces of DNA with the high efficiency of infection of lambda particles. The cos sites allow us to do that.

Question 8

What happens when the cosmid infects the E.coli cell?

Answer 8

The DNA recircularises via the cos site. The infection is a highly efficient process.

Question 9

How will the medium select for the E.coli cells that are infected with the cosmid?

Answer 9

Due to the ampicillin resistant gene, the E>coli cells containing the cosmid will be selected.

Question 10

Will the cosmids gve rise to bacterial colonies or plaques?

Answer 10

Colonies.

Question 11

What are cosmids used for?

Answer 11

Constructing genomic libraries.

Question 12

What is a genomic library?

Answer 12

A collection of cloned fragments that encompass the whole genome of an organism. It will contain all the genes of the organism and the non-coding regions as well.

Question 13

What is the equation for the number of clones needed?

Answer 13

N=ln(1-p)/ln(1-a/b) where N is the number of clones needed, P is the probability that the gene is present, a is the average size of DNA insert in vector and b is the total size of the genome.

Question 14

What are problems with the construction of a genomic library?

Answer 14

6-cutter enzmes such as BamHI cuts on average once every 4096 nucleotides. Many fragments will be too small , and some too large to clone. Sau3A (4 cutter) will produce fragments that are too small to clone.

Question 15

How can this problem with genomic libraries be solved?

Answer 15

Perform a partial digest with Sau3A (stop digestion going to completion and taking samples at various timepoints) and select sizes appropriate for cloning and clone into a BamHI-cut replacement vector.

Question 16

What are all the steps in making a genomic library?

Answer 16

Partial digest with Sau3A, ligate into a BamHI cut cosmid, package into lambda particles and infect E.coli (with high efficiency) and plate onto agar and ampicllin and recover the colonies.

Question 17

What are some methods to clone even larger fragments?

Answer 17

Bacterial artificial chromosomes (BACs) - based on F plasmid of E.coli and can clone fragments up to 300kb. Bacteriophage P1 that can clone up to 110kb and P1 derived artificial chromosomes (PACs) that are cosmid-like vectors based on P1 and can clone up to 300kb.

Question 18

What is cDNA cloning?

Answer 18

Copy or complementary cloning of DNA copies of eukaryotic mRNA.

Question 19

Why would cDNA cloning be done?

Answer 19

As eukaryotic genomes are large and most is non-coding, so this can allow cloning of coding regions alone without introns. It takes advantage of differential gene expression.

Question 20

How can mRNA be isolated?

Answer 20

Isolate the total RNA from the tissue of interest and purify it (using PolyA-RNA binding by oligo-dt chromatography) and use this as the substrate for cDNA snythesis.

Question 21

How can the mRNA be made into a double strand?

Answer 21

Using reverse transcriptase to synthesise a complementary DNA strand to the RNA template.

Question 22

Why does the mRNA need to be made into a double strand?

Answer 22

Restriction enzymes can only work on double stranded templates.

Question 23

Why do oligo(dT) primers need to be bound to the mRNA?

Answer 23

They can bind to the Poly(A) tails and act as a primer for the reverse transcriptase.

Question 24

What molecule is formed after reverse transcriptase has acted?

Answer 24

A DNA-RNA hybrid double stranded molecule.

Question 25

How can the RNA be partially degraded?

Answer 25

Ribonuclease H (RNase H) partially degrades the strand so that some RNA fragments are left base-paired to the DNA strand.

Question 26

What is the purpose of partially degrading the RNA strand?

Answer 26

The RNA fragments act as primers to DNA polyermase I. The proof reading capability of DNA pol I means RNA is replaced by DNA and a double stranded DNA molecule is formed.

Question 27

Why must linkers be added to cDNA ends?

Answer 27

cDNAs are blunt ended and are inefficient to clone.

Question 28

What are linkers?

Answer 28

Short, synthetic double-stranded oligonucleotides that contain a restriction site. They are used in such high concentration that despite their blunt ends they are ligated to the ends of the cDNA.

Question 29

What happens after the linkers are added to the cDNA?

Answer 29

It is digested with restriction enzymes that cut the restriction site in the linker so you end up with cDNAs with sticky ends.

Question 30

What is the total collection of cDNA clones produced called?

Answer 30

A cDNA library.

Question 31

What can be used to protect internal BamHI sites in the cDNA?

Answer 31

Methylase so internal DNA doesn’t get cut.