3 - Molecular Cloning Methods

Question 1

In what year was the first sheep cloned

Answer 1

1996 cloned but born 1997

Question 2

What three steps are involved with the cloning process?

Answer 2

Place a foreign gene into bacterial cells

Separate individual cells

Grow colonies from each of them.

Question 3

What year was the first cloning experiment?

Answer 3

1973

Question 4

Linn and Arber discovered restriction endonucleases in ____ in the late ____.

Answer 4

Linn and Arber discovered restriction endonucleases in E. coli in the late 1960.

Question 5

Why are Restriction endonucleases named that way?

Answer 5

They restrict the host range of virus

They cut at sites within the foreign DNA, rather than chewing it away at the ends.

Question 6

An Enzyme from ______ _______ Rd was the first enzyme to show specificity in cutting DNA.

Answer 6

An Enzyme from Haemophilus influenzaestrain Rd was the first enzyme to show specificity in cutting DNA.

Question 7

How are Restriction Endonucleases designated?

Answer 7

Restriction enzymes derive the first three letters of their names from the Latin name of the microorganism that produces them:
First letter is the first letter of the genus H

The next two letters are the first two of the species

In some cases the strain designation is included, in this case d

Question 8

Regarding Restriction Endonucleases, What is a rare cutter?

Answer 8

A restriction endonuclease that recognizes 8-bp(example NotI) therefore cut much less frequently

Question 9

What are Heteroschizomers?

Answer 9

Heteroschizomers (Greek: hetero = different; schizo = split) or neoschizomers are enzymes that recognize different sites in identical sequences

Question 10

What are Isoschizomers?

Answer 10

Isoschizomers (Greek: iso= equal) are enzymes that cut at the same site in the same sequence.

Question 11

What is a palindrome sequence?

Answer 11

A palindrome sequences is a sequence with twofold symmetry. They read the same forward and backward.

Question 12

What two types of cuts are possible with Restriction endonucleases?

Answer 12

Blunt - Flat cut leaving blunt ends

Single strand overhang - staggered cuts in the two DNA strands

Question 13

If endonucleases can cut invading DNA, why don’t they destroy the host cell’s own DNA?

Answer 13

Methylase enzymes recognize and methylate the same DNA sites. Once methylated, DNA sites are protected against most endonucleases.

Question 14

Almost all restriction enzymes are paired with a methylase enzyme, that recognizes and methylates the same DNA sites. what is this system called?

Answer 14

This system is called the R-M system (restriction-modification system)

Question 15

Restriction enzymes are classified biochemically into what three types?

Answer 15

Restriction enzymes are designated as Type I, Type II, Type III.

Question 16

A major type of Type II enzymes are sometimes referred to as ______ enzymes.

Answer 16

A major type of Type II enzymes are sometimes referred to as Type IV enzymes.

Question 17

Type I and III systems are unique how?

Answer 17

Type I and III systems, both the methylase and restriction activities are carried out by a single large enzyme complex.

Question 18

True or False:

Type I and III systems Cut adjacent to the recognition site

Answer 18

False

Type I and III enzymes recognize specific DNA sequences, but the sites of cleavage are at variable distances from the recognition sites, and can even be hundreds of bases away.

Question 19

What is required for all three classifications?

Answer 19

ATP

Question 20

How is Type II restriction enzymes unique?

Answer 20

In type II systems, the restriction enzyme is independent of its methylase, and cleavage occurs at very specific sites that are within or close to the recognition sequence.

Question 21

What of the three classifications is the most common? (Type I, II, or III)

Answer 21

The vast majority of known restriction enzymes are of type II.

Question 22

How are Type II enzymes further classified according to their recognition site?

Answer 22

Type II
Type IIa
Type IIb
Type IIs

Question 23

How do type II enzymes cut?

Answer 23

Most type II enzymes cut palindromic DNA sequences

Question 24

How do type IIa enzymes cut?

Answer 24

Type IIa enzymes recognize non-palindromic sequences and cleave outside of the recognition site

Question 25

How do type IIb enzymes cut?

Answer 25

Type IIb enzymes cut sequences twice at both sites outside the recognition sequence.

Question 26

How do type IIs enzymes cut?

Answer 26

Type IIs enzymes cleave the DNA at a considerable offset from the recognition sequence.

Question 27

All gene cloning experiments require carriers also called _____.

Answer 27

All gene cloning experiments require carriers also called vectors.

Question 28

All vectors fall into which two major classes?

Answer 28

• Plasmids

* Phages

Question 29

The first group of plasmids vectors to be developed are known as?

Answer 29

pBR plasmid series, They are no longer used but are an easy model to describe.

Question 30

How is it possible to find the clones that have received recombinant DNA?

Answer 30

The replicating plate technique. This is not a selection technique but a screening technique or process