Wright Lecture 4: Cloning Genes and their mRNA Transcripts

Question 1

Cloning by complementation

Answer 1

Requires mutant strain of bacterium or other host organism

ie. Inserting His+ gene plasmid into His- bacterium

Question 2

Inserting gene into E. coli (complementation)

Answer 2

1. Purify total DNA from WT E. coli (His+)
2. Cleave E. coli DNA with restriction endonuclease
3. Construct genomic library with E. coli DNA in a suitable cloning vector
4. Transform mutant strain of E. coli that lacks ability to synthesize histidine (His-)
5. Plate transformed bacteria -His medium 6. Only colonies that grow contain His biosynthetic pathway genes: select

Question 3

Hybridization

Answer 3

Can be used to identify specific DNA sequence in genomic library
Requires probe that is complementary or partially complementary to target cloned DNA sequence

Question 4

Detecting genes (hybridization)

Answer 4

1. Transfer bacterial colonies from medium to nitrocellulose filter
2. Lyse cells and denature DNA
3. Wash filter with unrelated single stranded DNA
4. Add radioactive nucleic acid probe
5. Some of the probe molecules become hybridized to the DNA of the bacteria
6. Visualize using X-ray film

Question 5

Non-coding regions of DNA

Answer 5

Intergenic regions (between genes
Introns
Repetitive DNA
(less than 2% of DNA codes for proteins)

Question 6

Satellite DNA

Answer 6

Very large arrays of tandemly repeating, non-coding DNA

Main component of functional centromeres

Question 7

Introns

Answer 7

Between protein coding axons
Spliced out in mRNA
Make up much of DNA in gene of eukaryotes

Question 8

RNA at a basic pH

Answer 8

2’-hydroxyl catalyzed hydrolysis of RNA

Question 9

2-hydroxyl catalyzed hydrolysis of RNA

Answer 9

1. Nucleophilic 2’ hydroxyl attacks adjacent phosphate
2. Cleavage of 5’ phosphate bond and formation of 2’,3’ cyclic phosphate modification
   Subsequent hydrolysis of intermediate provides and mixture of 2’ and 3’ monophosphates

Question 10

Central dogma of molecular biology

Answer 10

Pathways of information transfer within the cell

DNA (replication; Transcription; RNA; translation; protein

Question 11

David Baltimore

Answer 11

1975

Some viruses convert their RNA genome into DNA using reverse transcriptase during infection of cells

Question 12

Synthesis of of complementary DNA from purified mRNA

Answer 12

1. mRNA, anneal oligo(dT) primer (can be at Poly(A)tail)
2. Reverse transcriptase , dNTPS
3. RNaseH degrades mRNA and leaves primers for new synthesis
4. Synthesis and removal of primers using DNA polymerase I
5. DNA ligase joins sections of DNA
   Result: dsDNA

Question 13

cDNA

Answer 13

Derived from mRNA extracted from tissue

Can be cloned into plasmid vector

Question 14

Detect cDNA clone (using antibodies)

Answer 14

1. Transform bacteria cell with recombinant DNA plasmid
2. Grow on selective medium
3. Transfer individual colonies to microtiter dish to grow
4. Print colonies to membrane filter and grow on medium so cDNA protein product is expressed
5. Lyse cells in situ (protein produce will adhere to membrane)
6. React protein on filter with radioactive antibody, wash off excess antibody
7. Identify clone through visualizing with autoradiography