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AMB > LECTURE 2 (The toolkit) > Flashcards

LECTURE 2 (The toolkit) Flashcards

1
Q

Characteristics of endonucleases type II (main ones) (6)

A
  • conventional turnover
  • recognize site with rotational symmetry
  • Require Mg2+ as a cofactor
  • separate methylase and endonuclease
  • catalyze the hydrolysis of DNA
  • normally cut dsDNA but there’re exceptions!
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2
Q

How were endonucleases first discovered? Why are they called “Restriction Enzymes”?

A

They were found in bacteriophage replication. It’s an enzyme that cuts DNA at a specific place (restricting the rest).

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3
Q

What is the recognition or restriction site?

A

specific sequence where the endonuclease cuts.

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4
Q

Why do they separate methylase and endonuclease? What are these two enzymes?

A

endonuclease (cuts DNA or RNA)
methylase (methylate the substrates, mainly at the end)
Methylation makes DNA resistant to RE and endonucleases cut DNA. RE want to separate these two so they have a free way to cut at the specific site bc the DNA is not degraded by an endonuclease nor methylated.

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5
Q

Characteristics EcoRI.(6)

A
  • discovered in E.coli.
  • Produces a 5’ overhang
  • p group attached to the 5’ end
  • Recognizes 6bp seq
  • hydrolyses bt the 1st and 2nd bp
  • structure GAATTC (palindromic)
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6
Q

Characteristics PstI. (6)

A
  • Providencia stuartii
  • Produces a 3’ overhang
  • p group attached to 5’ end
  • Recognizes 6bp seq
  • hydrolyses bt the 5th and the 6th
  • structure: CTGCAG (palindromic)
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7
Q

Characteristics HaeIII (6)

A
  • Haemophilius aegyptius
  • Produces a blunt end
  • p group attached as well
  • Recognizes 4bp seq
  • hydrolyses bt 3rd and 2nd
  • Structure GGCG
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8
Q

Name other commercially available RE.

A

Hhal (4bp recognition)
HindIII(cuts after the 1st base, Haemophilius influenza)
NotI (8bp recognition, sticky ends)
Sau3A (GATC , 1/256 bases)
BglII (recognizes a discontinuous sequence, half sites are separated)

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9
Q

The most common RE are type II, and then?

A

type II S. recognize continuous (symmetric sequences) and asymmetric sequences.
type II G. bigger ones

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10
Q

What is an sticky end?

A

an overhang

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11
Q

Special case of BamHI and BgIII

A

Their sticky ends can be ligated respectively although they recognize different sequences. Restriction site is not retained.
imp: blunt ends can be ligated to any blunt ends

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12
Q

isoschizomers?

A

two RE that recognize the same sequence although they cut at different positions (aka neoschizomers)

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13
Q

isocaudomers?

A

don’t recognize the same seq but produce the same overhang.

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14
Q

What are ligases?

A
  • catalyze the ligation of DNA or RNA molecules
  • the molecules it ligates must be phosphorylated at the 5’ end (at least 1 strand)
  • They require ATP + cofactor + very high [blunt ends] because it’s less efficient with blunt ends
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15
Q

Example of ligase.

A

bacteriophage T4 ligase. encoded by a bacteriophage.

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16
Q

What are kinases? (4)

A
  • catalyze the addition of p (from ATP) to the 5’ end of RNA or DNA
  • Mg2+ required as a cofactor
  • ATP is necessary
  • They can label NA
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17
Q

What is alpha32p?

A

phosphate that was used for radiolabelling but it is not used anymore.

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18
Q

What are alkaline phosphatases?

A
  • catalyze the opposite reaction to kinases
  • they remove the p group from the 5’ end
  • they were extracted from eukaryotic sources like calf intestine (very thermostable) and shrips (SAP) –> very heat unstable bc they live in cold environment.
  • dephosphorylation is of great use in cloning procedures
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19
Q

What are polymerases?

A
  • catalyze the formation of a 2nd strand of DNA from a template.
  • catalyze the addition of deoxinucleotides (dNTPs)
  • it requires Mg2+ cofactor
  • They can have exonucleotic activity (degrade DNA) e.g the holoenzyme DNAP I
20
Q

Properties of DNAP I (E.coli, holoenzyme) (4)

A
  • 109 kD
  • 1 subunit
  • pH optimum 7.4
  • RNAse H activity
21
Q

Properties of Klenow fragment

A
  • 76kD
  • 1 subunit
  • pH optimum 7.4
  • no RNAse H activity
22
Q

Properties Taq DNAP (3)

A
  • 94 kD
  • 1 subunit
  • pH optimum 8.3
23
Q

Properties T7 DNAP

A
  • 92 kD
  • 2 subunits
  • 7.6
24
Q

Reverse transcriptase. Give an example of RT in humans.

A

it’s a polymerase that uses RNA as a template. They can also have exonuclease activity e.g RNAse H
Telomerase acts as a RT in telomeres to elongate them

25
Q

RNAP?

A

used to make RNA. e.g T7 RNAP (requires ATP)

encoded by bacteriophages.

26
Q

Which are the three enzymes encoded by bacteriophages?

A

RNAP, kinases and ligases.

27
Q

What are non-specific nucleases? give 3 examples of RE that are non-sequence specific.

A

Exonucleases and endonucleases (cut DNA and RNA) used by bacteria to defend themselves from viral attack
We can also find some RE that are non-specific
e.g Bal 31
e.g S1 (recognizes only ss DNA or ss regions)
e.g DNAsel

28
Q

What is RNAse H ?

A

Exonuclease activity: RNAse H (H–> heteroduplex, RNA-DNA heteroduplex, RNA is degraded and you end up with just DNA)

29
Q

What requires Mg2+ as a cofactor?

A
  • Restriction enzymes
  • Kinases
  • Polymerases
30
Q

What is a gel electrophoresis? How does it work?

A

The movement of molecules in an electric field in order to analyze the proteins and NA. DNA is -vely charged therefore it’s gonna move towards the +vely charged electrode (red). electrophoresis a.k.a horizontal submarine

31
Q

What is the substrate through which DNA migrates in a gel electrophoresis?

A

Agarose (for big molecules) or polyacrilamide(for smaller molecules)

32
Q

What is the buffer used in electrophoresis?

A

TBE: tris boric acid, edta + water

33
Q

what does the speed of migration depends on? (4)

A

[agarose], size, mass and shape and NOT in the amount of charge

34
Q

What is the preparative gel electrophoresis?

A

the staining of the molecules to visualize them, usually with Sybr green (previously ethidium bromide was used but it’s too toxic).

35
Q

What is analytical gel electrophoresis?

A

the estimation of the size using biological markers of previously known size e.g DNA of lambda phage.

36
Q

What are for the 4 different types of blotting?

A

Southern: separates DNA
Northern: separates RNA
Western: separates proteins
South-Western: separates proteins binding to DNA

37
Q

How does blotting work?

A

Transfer the DNA from electrophoresis into a nitrocellulose or nylon membrane. Then the specific seq can be analyzed by hybridizingg the fluorescent probe(piece of DNA that matches our seq of interest) that makes the section we’re interested in; fluorescent signal.

38
Q

What is liquid phase hybridization? What two processes occur via this?

A

Occurs in liquid. PCR and sequencing.

39
Q

What are Ribonuclease protection Assays? and DNAse?

A

Analyze RNA in a solution by adding fluorescent probes : the probes are ss and need to be antisense. Allow bindings by heating the mixture and then cooling down. The rubonucleases will degrade the ssRNA and the ds molecules remain. the gel shows different seq of different lengths. this is not very much used. from the size of the band you can estimate how much of the original NA was present.
The same can be done with DNA in DNAse protection Assays.

40
Q

Difference between blotting and protection assays

A

In protection assays, the stuff that’s not hybridized is destroyed by ribonucleases unlike in blotting.

41
Q

What is gel electrophoresis for? (3)

A

Finding the mass, size and concentration of DNA, RNA and proteins

42
Q

What if RE are not used with the appropriate buffer?

A

They won’t be active and they’d cut in a unpredictable manner.

43
Q

RE in nature, what do they do?

A

Limit the growth of bacteriophages and have host-specific methylases that avoids the host (bact) from digesting their own NA.

44
Q

What requires ATP?

hint: the same ones that are encoded by bacteriophages

A
  • RNA polymerases (not DNA)
  • Ligases
  • kinases
45
Q

Are ribonucleases found in the env? where?

A

yes in human skin

46
Q

Are ribonucleases easy or difficult to remove?

A

They’re difficult to remove from cloning procedures.

AMB (12 decks)

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