9.2 Cloning DNA fragments

Question 1

What is molecular cloning?

Answer 1

This type of cloning purifies a specific DNA fragment away from all other fragments and makes many identical copies of the fragment.

Question 2

The group of replicated DNA molecules is known as a?

Answer 2

DNA clone

Question 2

What are the two basic steps of molecular cloning?

Answer 2

1. Insert DNA fragments into cloning vectors to make a recombinant DNA molecule.
2. Transport recombinant DNA into living cell to be copied.

Question 3

What do we use plasmid cloning vectors for?

Answer 3

to create recombinant DNA molecules

Question 4

What are the three main features of Plasmid cloning vectors?

Answer 4

1. An origin of replication
2. Restriction site(s) for cloning insert DNA
3. A selectable marker (e.g. antibiotic resistance)

Question 5

What two types of artificial chromosomes are alternate cloning vectors that can carry large inserts?

Answer 5

Bacterial and yeast

Question 6

Explain how a recombinant DNA molecule is created

Answer 6

from the cutting and ligating together of vector and inserted fragment

Question 7

The simplest vectors are small circles of double-stranded DNA known as __________

Answer 7

plasmids

Question 8

(step 1 of molecular cloning) Creating recombinant DNA molecules with plasmid vectors:

Answer 8

1. Digestion of the plasmid vector and human genomic DNA with a restriction enzyme results in complementary sticky ends
2. DNA ligase is used to seal the phosphodiester backbones between vector and insert

Question 9

(step 2 of molecular cloning) Host cells take up and amplify recombinant DNA:

Answer 9

1. Scientists first add recombinant DNA molecules to a suspension of E.coli sensitive to the antibiotic ampicillin. (only 0.1% of cells will be transformed with plasmid. Only the cells with recombinant plasmid will grow on media with ampicillin)
2. Under certain conditions such as the technique of electroporation, the plasmids will enter about 1 in 1000 cells. (these protocols increase the permeability of the bacterial membrane, in essence punching holes through which the DNA gains entry).
3. Each cell multiplies to produce millions of genetically identical cells, each with a recombinant plasmid.

Question 10

Define transforming

Answer 10

The process by which a cell or organism takes up foreign DNA

Question 10

What is the probability that any one plasmid will enter any cell?

Answer 10

0.0001

Question 10

What is genomic equivalent?

Answer 10

it is the number of clones in a perfect library

Question 10

A perfect genomic library has one copy of every sequence in the _____________

Answer 10

entire genome

Question 10

What is the formula for calculating the number of clones in a perfect library (impossible to obtain a perfect library)?

Answer 10

length of genome/average size of inserts

Question 10

What is a genomic library

Answer 10

Long-lived collection of cellular clones that contains copies of every sequence in the whole genome inserted into a suitable vector.

Question 10

How many genomic equivalents do genomic libraries usually have?

Answer 10

4-5 (gives an average of 4-5 clones for each locus, 95% probability that each locus is present at least once)