Lecture 4 - Cloning Genes and their mRNA Transcripts

Question 1

How are specific DNA (gene) sequences identified?

Answer 1

complementation and hybridization

Question 2

What is the genome?

Answer 2

the total amount of DNA from both the nucleus and the mitochondria

Question 3

What is each colony derived from?

Answer 3

a single bacterial cell with a single fragment of the genome

Question 4

What does cloning by complementation require and what is the limitation of this technique?

Answer 4

• requires a mutant strain of the bacterium, or other host organism
• the limitation is that you NEED a mutant strain (for cloning particular types of genes)

Question 5

What is an example of cloning by complementation?

Answer 5

the cloning of the histidine biosynthetic genes from E. coli

Question 6

Which cells cannot synthesize histidine and therefore cannot grow on minimal medium in a E. coli host cell?

Answer 6

A chromosome in an E. coli host cell with a mutation on the His- biosynthetic genes

Question 7

What needs to be introduced to the E. coli cells in order for them to be able to grow on minimal medium?

Answer 7

• a plasmid carrying a copy of the His+ biosynthetic genes that enable the synthesis of histidine

Question 8

What does the identification of a specific DNA sequence in the genomic library by hybridization require?

Answer 8

A hybridization probe that is complementary, or partially complimentary, to the targeted cloned DNA (gene) sequence

Question 9

What kind of DNA binds to the nitrocellulose?

Answer 9

single-stranded DNA

Question 10

What characterizes the complexity of eukaryotic genomes?

Answer 10

• intergenic regioins (DNA between genes)
• introns
• repetitive DNA (satelite DNA at centromeres, telomeres, microsatelite and ministelite DNAs dispersed throughout the genome)
• DNA coding for protein, for example, is

Question 11

What nuclease will denature with heat?

Answer 11

deoxyribose

Question 12

What nuclease will denature with heat, but then will grow back again (renature) and it hard to kill the enzymes of?

Answer 12

ribose nuclease (RNA)

Question 13

What is converted simultaneously into different complimentary DNA strands?

Answer 13

many different mRNAs that are each coding for different proteins

Question 14

What is used as primer for new DNA synthesis?

Answer 14

degraded RNA fragment

Question 15

What synthesizes new DNA strand in segments and removes RNA primers?

Answer 15

DNA polymerase I

Question 16

What joins DNA fragments?

Answer 16

DNA ligase

Question 17

What is derived from many mRNAs extracted from a given tissue, and then are cloned into a plasmid vector?

Answer 17

cDNAs

Question 18

What is the purpose of detection vectors?

Answer 18

to detect a cDNA clone coding for a specific protein using antibodies

Question 19

What are the cycles of the Polymerase Chain Reaction (PCR)?

Answer 19

1) denaturation of the DNA
2) primer annealing
3) strand elongation