Lecture 2

Question 1

Restriction Endonucleases (REs)

Answer 1

cleave double stranded DNA by breaking phosphodiester bonds

Question 2

What is the role of restriction endonucleases in nature?

Answer 2

protect cell from infection by foreign DNA

ex: bacteriophage (virus)

Question 3

What is the type of Restriction enzymes we use and describe?

Answer 3

Type II:

• smaller usually homodimers
• only require Mg++ for activity
• unlike type 1, cleave DNA at specific sites
• *are used for cloning

Question 4

Restriction sites

Answer 4

• recognition sequence on DNA that restriction enzyme binds to and cleaves the phosphodiester bond
• usually palindromic (read the same 5’ to 3’ on both strands), but the sites can vary both in nucleotide sequence and length
• Different enzymes recognized distinct sites

Question 5

Restriction digestion (or digest)

Answer 5

in cloning, adding a specific restriction enzyme (or enzymes) to specific DNA to obtain predictable or inter predictable DNA fragments

Question 6

Blunt ends

Answer 6

a cut at the center of dyad= blunt ends

NNNGG/ CCNNN
NNNCC/ GGNNN

Question 7

Sticky (cohesive) ends

Answer 7

a cut left of center produces cohesive ends with 5’ overhangs or to right of center, 3’ overhangs

Question 8

How do you determine the frequency of a restriction site?

Answer 8

frequency of cleavage sites in DNA: 1/4^n where n=length of restriction site

4bp site- frequent cutter
8bp site- very infrequent

Question 9

Restriction enzyme nomenclature

Answer 9

first letter= first initial of genus from which the enzyme was isolated

second and third letters= first two initials of species (first three letters are italicized)

Fourth letter(optional)= is letter of strain, not italicized

Question 10

Perfect isoschizomers

Answer 10

enzymes that recognize the SAME DNA SEQUENCE (and cut the same), but are isolated from different organism

Question 11

Imperfect isoschizomers

Answer 11

enzymes that recognize the same DNA sequence, but one enzyme recognized UNMETHYLATED DNA and one restricts only methylate DNA.

Question 12

Neoschizomers

Answer 12

enzymes that recognize the same sequence, but CUT DIFFERENT NUCLEOTIDES

Question 13

When setting up a RE digestion reaction: how do you determine what buffer to use?

Answer 13

• buffers are supplied at 10X with the enzyme
• some enzymes also require the presence of BSA(now it comes already spikes in the buffer)
• if performing a double digest, look at each enzymes activity in the various buffers, or look at chart at neb.com

Question 14

Enzyme activity (unit)

Answer 14

the amount of enzyme required to digest 1 microgram of DNA in a 50 uL reaction in 1 hr

Question 15

When setting up a RE digestion reaction: how do you determine how much enzyme you should add to your reaction?

Answer 15

• usually add 5 to 10 fold excess than needed
• NEVER let volume of the total enzyme exceed 10% reaction volume (adding to much enzyme can lead to star activity-ex: cutting sequence other than recognition site)
• ALWAYS add last to your reaction

Question 16

What is a expression vector and what are some of its basic features?

Answer 16

-have basic features in common with cloning vectors, but also carry components necessary to transcribe and translate your gene of interest

Basic features:

• origin of replication- often lower copy number than cloning vector
• selectable marker (ex: antibiotic resistance)
• Multiple cloning sites

usually no screenable marker, because these are rarely compatible with expression of the inserted gene

• promoter- drives transcription of gene
• Ribosome binding site-present near start of transcript and signals ribosome to bind and scan for start codon
• start codon- nucleotide triplet (codon) that signals start of translation
• multiple cloning site- désigned to allow insertion in a predictable reading frame and orientation
• fusion tag- can aid in purification, detection, etc of protein- not always desired/ present because it changes the protein.

Question 17

What does a restriction endonuclease digestion do?

Answer 17

breaks a phosphodiester bond

Question 18

DNA ligase

Answer 18

creates a phosphodiester bond

catalyzes covalent bonds formation between 3’OH and 5’ PO4 on DNA

Question 19

Blunt ends characteristics

Answer 19

• any two blunt ends can be ligated

- less efficient, but just give it a little more time

Question 20

Sticky ends characteristics

Answer 20

• very efficient in proper buffer conditions

- Must be compatible (have complimentary base pairs) in over to be ligated together

Question 21

How are compatible cohesive ends produced?

Answer 21

produced by restriction enzymes that recognize different sites, but creates staggered ends that are complementary

Question 22

Can hybrid sites that are formed by ligation be recleaved?

Answer 22

NO

Question 23

During ligation with insert, plasmid vectors have a tendency to relegate upon themselves (to generate original circular plasmid) rather than with an insert. How can we minimize this?

Answer 23

pohophatase treatment

Forced cloning

Question 24

Does ligation occur spontaneously?

Answer 24

No, ligation only occurs in the presence of an enzyme such as ligase, it does NOT OCCUR SPONTANEOUSLY

Question 25

Phosphatase treatment

Answer 25

- useful if cloning with only one enzyme and in blunt-end cloning - treat ONLY the vector with alkaline phosphatase (AP) - AP removes 5' phosphates from ends of DNA - vector cannot relegate upon itself; it can only ligate to insert - possible disadvantages: insert can go in either direction - MUST heat inactivate AP before ligation!! - after ligation, each strand will have a nick reparable in cells

Question 26

Forced (directional) cloning

Answer 26

- Double digestion with two different enzymes that leave incompatible ends will prevent vector from re-ligating (if stuffer fragment is removed) - Insert is cut with same two enzymes use to cut the vector - Another advantage is that the orientation of the insert is forced

Question 27

In our project what will our insert and vector DNA be digested with?

Answer 27

NcoI and NotI restriciton enzymes

Question 28

How does DNA move on a gel?

Answer 28

DNA is negatively charged, so it moves toward the anode. The agarose gel matrix retards large molecules more than small, so small fragments move a further distance through the gel.

Question 29

Gel Red

Answer 29

- will be used instead of ethidium bromide for the visualization of DNA under UV light - Gel red binds specifically to DNA and is excited by UV light; therefore when the gel is exposed to UV light you can visualize the DNA

Question 30

DNA loading buffer

Answer 30

also called loading dye or sample buffer - is added to DNA sample before loading the gel - contains glycerol which is heavier than TBE or TAE buffers - contains ore or more dyes in it