Lab 6

Question 1

what are restriction endonucleases?

Answer 1

Restriction endonucleases are used as molecular scalpels to cut DNA in a precise and predictable manner.

Question 2

what are nucleases?

Answer 2

display the general property of breaking the phosphodiester bonds that link adjacent nucleotides in DNA and RNA molecules

Question 3

what are exonuclease?

Answer 3

progressively digest from the ends of nucleic acid molecules.

Question 4

what are endonucleases?

Answer 4

cleave nucleic acids at internal positions,

Question 5

where is the term restriction enzyme derived from?

Answer 5

The term restriction endonuclease is derived from the observation that certain bacteria can block bacteriophage infections by specifically destroying the incoming bacteriophage DNA. Such bacteria are known as restricting hosts since they restrict the expression of foreign DNA.

Question 6

how is the DNA of the host cell protected from restriction enzymes?

Answer 6

The DNA of the host cell itself is protected from the nucleases because host bases are methylated at the sites recognised by the endonuclease, therefore, the nucleases cannot catalyse hydrolysis of the modified substrate (host DNA). Any DNA that enters the cell and is not methylated at those specific sites is subject to cleavage by restriction endonucleases.

Question 7

what are the three classes of endonuclease?

Answer 7

Type I, Type II and Type III

Question 8

which type of endonucleases are used in DNA?

Answer 8

type II

Question 9

Why are type 2 restriction enzymes useful?

Answer 9

Each has only restriction activity.

Each cuts in a predictable and consistent manner, at a site within or adjacent to the
recognition sequence.

They require only the magnesium ion (Mg2+) as a cofactor; ATP is not required

Question 10

how do restriction enzymes work?

Answer 10

scans DNA molecule, stopping only when it recognizes a specific sequence of nucleotides

Question 11

how long are the restriction endonuclease recognition sites? what do they look like?

Answer 11

4-8 base pairs. they’re also palindromic

Question 12

what does palindromic mean?

Answer 12

This means that the recognition sequence read forward on one DNA strand is identical to the sequence read in the opposite direction on the complementary strand. Put another way, the 5’ to 3’ sequence is identical on each DNA strand.

Question 13

explain the process of the restriction enzyme cutting the DNA strand

Answer 13

Within or very near the recognition site, the restriction enzyme catalyses a hydrolysis reaction that uses water to break a specific phosphodiester linkage on each strand of the DNA helix. Two DNA fragments are produced, each with a phosphate group at the 5’ end and a hydroxyl group at the 3’ end.

Question 14

what are the two types of cuts done by restriction enzyme?

Answer 14

-these flush-or-blunt ended fragments (cuts right through the double strand)

-Other endonucleases cleave each strand off-centre in the recognition site, at positions two to four nucleotides apart

Question 15

which endonucleases cleave right through and leave blunt ended fragments

Answer 15

HaeIII

Question 16

which endonucleases leave overhangs?

Answer 16

EcoRI, BamHI, and HindIII

Question 17

why are sticky ends useful to making recombinant DNA

Answer 17

These exposed nucleotides serve as a template for realignment, allowing the complementary nucleotides of two like restriction fragments to hydrogen bond to one another. A given restriction enzyme cuts all DNA in exactly the same fashion, regardless of whether the source is a bacterium, a plant, or a human. Thus, any sticky- ended fragment can be recombined with any other fragment generated by the same restriction enzyme.

Question 18

why can the sticky ends of fragments cut with the same restriction enzyme recombine with each other?

Answer 18

cause it cuts at a specific sequence, and they all have the same sequence, so u can recombine them with each other

Question 19

what does the size of the DNA fragments generated depend on

Answer 19

The size of the DNA fragments generated depends on the distance between recognition sites

Question 20

what gives us a greater chance that a given restriction site will occur?

Answer 20

a longer DNA molecule

Question 21

how many restriction sites do BamHI EcoRI HindIII have?

Answer 21

BamHI: 5
EcoRI: 5
HindIII: 7

Question 22

what is the goal of this lab.

Answer 22

In this experiment, three samples of purified DNA from λ-bacteriophage (48,502 base pairs in length) will be incubated with one of three restriction enzymes: EcoRI, BamHI, and HindIII. Each of the enzymes has five or more restriction sites in the bacteriophage DNA and therefore produces six or more restriction fragments of varying lengths. A fourth control sample will be incubated without a restriction enzyme and should remain intact. The digested DNA samples will then be loaded and run in a 0.8% agarose gel.we want to determine the base pair sizes of the DNA cut with ECOri and BAMHI by basing ourselves off of the HINDII graph (given to us)

Question 23

how will we visualize the DNA fragments after running them in the agarose gel?

Answer 23

The characteristic pattern of bands produced by each restriction enzyme will be visualized by staining the samples with methylene blue, a dye that binds to the DNA molecules.

Question 24

what is a doublet?

Answer 24

Two fragments of similar size will often produce a single heavy band referred to as a doublet. Doublets will appear brighter than single bands on the agarose gel

Question 25

what is the link between migration distance and base pair length?

Answer 25

bigger fragments (more base pairs) migrate less far

Question 26

how is the number of base pairs expressed?

Answer 26

in kilobase-pair size

Question 27

how are we finding the base pair sizes for EcoRI and BamHI?

Answer 27

we are making a graph based on our HindII values (given to us). base-pair length (y-axis) versus the distance migrated in cm (x-axis) are the values. we are then plugging in our migration distances found from our agarose gel electrophoresis and assigning them to a base pair size, as this is uniform throughout all dna fragments

Question 28

What is the recipe for making a 0.8% agarose gel?

Answer 28

0.4 g of agarose to 50 ml of 1X TAE buffer in a 250 ml Erlenmeyer flask.

Question 29

where are restriction enzymes found?

Answer 29

Part of the bacterial immune system

Question 30

how many fragments and sizes would be formed if lambda DNA was circular, the two ends of the DNA are connected, and it was cut with BamHI?

Answer 30

4 fragments