Wright 4 - Cloning Genes and their mRNA transcripts

Question 1

What are two methods to identify a specific DNA sequence (genes) by?

Answer 1

• Complementation

- Hybridization

Question 2

What are the 6 steps of cloning by complementation?

Answer 2

1. Purify total DNA from a wild-type strain of E. coli.
2. Cleave E. coli. DNA with a restriction endonuclease
3. Construct a genomic library with the E. coli DNA in a suitable cloning vector
4. Transform a mutant strain of E. coli that lacks the ability to synthesize histidine, ie is a His- mutant
5. Plate transformed bacterial mutant cells on minimal medium lacking histidine. Only cells with functional histidine biosynthetic genes will grow on this minimal medium
6. Colonies of cell that grow on the plate are selected as they contain the cloned histidine biosynthetic genes

Question 3

What type of cloning requires a mutant strain of the bacterium or other host organism from the outset?

Answer 3

Cloning by complementation

A plasmid with the lacking biosynthetic gene (and whatever other DNA is desired) is introduced.

Question 4

How can inserted DNA sequences be identified by hybridization?

Answer 4

Bacterial colonies spread out on a petri dish are transfered onto nitrocellulose filter. Then the cells are lysed and DNA denatured. The ssDNA is radioactively labeled with nucleic acid probes that are complementary to the DNA sequences that need to be identified. These become hybridized and exposure to x-ray film creates a autoradiograph. Spots on the x-ray film show the colonies where the DNA has been inserted

Question 5

What makes eukaryotic genomes complex?

Answer 5

• Intergenic regions (DNA between genes)
• Intron
• Repetitive DNA (eg. satellite DNA, telomeres, microsatellite and minisatellite DNA)

Only around 2% of DNA codes for protein

Question 6

What makes RNA unstable at basic pH?

Answer 6

The 2’ hydroxyl group can nucleophilically attack the 3’ phosphate group. This cleaves the phosphodiester bond and creates a mixture of 2’ and 3’ monophosphates

Question 7

What does the enzyme reverse transcriptase do in some viruses?

Answer 7

Convertes a RNA genome into DNA during infection of cells.

Question 8

How does reverse transcriptase convert ssRNA to dsDNA?

Answer 8

1. An oligo(dT) primer is annealed to the poly(A) tail of a strand of mRNA
2. Reverse transcriptase binds dNTPs (DNA nucleotides) to an mRNA template
3. RNAse degrades the RNA template, eaving only a few RNA fragments to act as primers for new DNA synthesis
4. DNA polymerase I synthesizes new DNA strands in segments and removes RNA primers
5. DNA fragments are joined by DNA ligase to make cDNA

This cDNA can be inserted into a cloning vector plasmid, usually ones containing a gene for resistance to an antibiotic

Question 9

What can be done with cDNA derived from mRNA (using reverse transcriptase).

Answer 9

cDNAs derived from the mRNA are extracted from a given tissue and cloned into a plasmid vector

Question 10

What are expression vectors?

Answer 10

They allow detection of a cDNA (cloned DNA, mRNA to DNA by reverse transcriptase) that has successfully been inserted into a transformed host. The expression vector is part of the inserted plasmid that can code for something like antibiotic resistance or a necessary biosynthetic product for selective growth medium