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BIT 410 > Lecture 2 > Flashcards

Lecture 2 Flashcards

1
Q

Restriction Endonucleases (REs)

A

cleave double stranded DNA by breaking phosphodiester bonds

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2
Q

What is the role of restriction endonucleases in nature?

A

protect cell from infection by foreign DNA

ex: bacteriophage (virus)

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3
Q

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

A

Type II:

  • smaller usually homodimers
  • only require Mg++ for activity
  • unlike type 1, cleave DNA at specific sites
  • *are used for cloning
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4
Q

Restriction sites

A
  • 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
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5
Q

Restriction digestion (or digest)

A

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

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6
Q

Blunt ends

A

a cut at the center of dyad= blunt ends

NNNGG/ CCNNN
NNNCC/ GGNNN

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7
Q

Sticky (cohesive) ends

A

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

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8
Q

How do you determine the frequency of a restriction site?

A

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

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

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9
Q

Restriction enzyme nomenclature

A

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

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10
Q

Perfect isoschizomers

A

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

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11
Q

Imperfect isoschizomers

A

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

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12
Q

Neoschizomers

A

enzymes that recognize the same sequence, but CUT DIFFERENT NUCLEOTIDES

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13
Q

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

A
  • 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
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14
Q

Enzyme activity (unit)

A

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

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15
Q

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

A
  • 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
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16
Q

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

A

-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.
17
Q

What does a restriction endonuclease digestion do?

A

breaks a phosphodiester bond

18
Q

DNA ligase

A

creates a phosphodiester bond

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

19
Q

Blunt ends characteristics

A
  • any two blunt ends can be ligated

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

20
Q

Sticky ends characteristics

A
  • very efficient in proper buffer conditions

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

21
Q

How are compatible cohesive ends produced?

A

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

22
Q

Can hybrid sites that are formed by ligation be recleaved?

A

NO

23
Q

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?

A

pohophatase treatment

Forced cloning

24
Q

Does ligation occur spontaneously?

A

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

25
Q

Phosphatase treatment

A
  • 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
26
Q

Forced (directional) cloning

A
  • 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
27
Q

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

A

NcoI and NotI restriciton enzymes

28
Q

How does DNA move on a gel?

A

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.

29
Q

Gel Red

A
  • 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
30
Q

DNA loading buffer

A

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

BIT 410 (8 decks)

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