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Molecular Cloning Flashcards

1
Q

( ! ) Why is Molecular Cloning Important?

Hint: PODPEER

A

P - Purify & Amplify Frags

O - Obtain sequences

D - Determine Gene Struc & Reg

P - Perform Site-Directed MGenesis

E - Express & Purify protein; BIOCHEM and STRUCTURAL analysis

E - Enable genome analysis with OVERLAPPING CLONES

R - Reintroduce (transgenesis)

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2
Q

What do Type II REs do?

A

Cleave DNA at specific bases

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3
Q

What do Type II Methylases do?

A

Methylate DNA at specific bases

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4
Q

What do DNA Polymerases do?

A

Copy DNA from a PRIMER 3’ end

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5
Q

What do RNA Polymerases do?

A

Make an RNA copy from DNA from a promoter

Opp. of Reverse Transcriptase

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6
Q

What do Reverse Transcriptases do?

A

Make a DNA copy of RNA from PRIMER 3’ end

Opp. of RNA Polymerase

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7
Q

What do DNA Ligases do?

A

Join two DNA molecules//fragments

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8
Q

What do Exonucleases do?

A

Remove NTs from ends of DNA

Sort of Opp. of Terminal Transferases

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9
Q

What do Terminal Transferases do?

A

Add a homopolymer tail to the ends of DNA

Sort of Opp. of Exonucleases

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10
Q

What do Polynucleotide Kinases do?

A

Add a PHOSPHATE to the 3’ ends of DNA

Opp. of alkaline phosphatase

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11
Q

What do Alkaline Phosphatases do?

A

Remove terminal phosphates from DNA ends

Opp. of Polynucleotide Kinases

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12
Q

What do Type II REs recognise, and what do they Require?

A

Recognise SHORT seq -
4-8nts

Require Mg+
(and no ATP)

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13
Q

What blocks RE cleavage of DNA?

A

Methylation!

  1. Added by the cognate methylase
    (Fully OR Hemimethylated)
  2. Dam + Dcm Methylation
    (Second A Or C)
    No cleavage if Meth site overlaps Restriction Site
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14
Q

What is the primary epigenetic signal on Prokaryotes?

A

Methylation

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15
Q

What will Dpn I only cleave?

A

METHYLATED DNA

Sequence GATC

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16
Q

What will Xho I not cleave?

A

EUKARYOTIC methylation @ CpG islands

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17
Q

What will Mbo I only cleave?

A

NONMETHYLATED DNA

18
Q

What does Ligase outcome depend on?

A

Conc of DNA

(Higher = InterMol + linear)

(Lower = IntraMol + circular)

19
Q

What 3 activities do many DNA Polymerases have?

A
  1. Extend 3’ end (standard)
  2. 3’ to 5’ Exonuclease
    (Proof-Reading)
  3. 5’ to 3’ Exonuclease
    (Remove DNA ahead)
20
Q

What is DNA Pol PROCESSIVITY?

A

Amount of time the Pol stays on the DNA replicating

21
Q

Why is T4 DNA Pol useful?

A

Trimming Fragment Ends -

Has high 3 to 5 EXONUCLEASE activity

22
Q

Why is T7 DNA Pol useful?

A

Chain Terminator SEQUENCING

Very high processivity

23
Q

Why is DNA Pol ONE useful?

A

Primer Removal (5 to 3)

24
Q

Why is Reverse Transcriptase useful?

A

Used to make cDNA from RNA template

25
Why are thermostable polymerases useful?
Used in PCR | Taq, Vent, Pfu
26
What is the Kelnow fragment of DNA Pol ONE?
DNA Pol without Exonuclease activity (5 to 3) Use to synthesis oligos for radiolabelling or Chain Terminator Sequencing
27
Why is T4 PN Kinase useful?
It transfers a P to the 5’ OH end LABELS that end (For fragments or oligo probes)
28
What are the FOUR properties of cloning vectors? (A MUM)
A - Autonomous Replication M - Marker (to select for cells) U - Unique Restriction Sites M - Minimal nonessential DNA (maximise cloning size range)
29
How long are PCR Primers?
17-25nt
30
What is the temperature progression in PCR?
95 > 55 > 72
31
What three things happen at the different temperatures in PCR?
Melt > Primer Anneal > Extension
32
What is PCR Error Rate like?
Greater than DNA rep Depends on polymerase proofreading Low error needed for cloning
33
How can you join ends cut by different REs??
Polymerase then Ligase Linker ligation Adapter ligation Add sequences to 5’ end by PCR
34
What is a Linker?
A DS oligo with a restriction site
35
What are Adapters?
Pre-formed cohesive ends (blunt and sticky end) Add by blunt ligation
36
What is a cos site?
The sites cleaved from the thread replicated from a rolling circle, which produce cohesive ends and are packaged in the phage
37
How do you make a Lambda insertion vector?
Remove inessential (lysogenic) genes up to 10kb and insert sequences To increase size - stuffer region with RE sites, package genome, remove stuffer new size up to 23kb
38
What is a contig?
All overlapping clones formed from chromosome walking
39
How do you screen PHAGE Genomic libraries?
96 well plating OR radioactive probes for gene
40
How do you screen BAC genomic libraries?
96 well plates Or UCSC Genome Browser

Molecular Genetics (5 decks)

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