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Molecular Genetics > Molecular Cloning > Flashcards

Molecular Cloning Flashcards

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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
Q

Why are thermostable polymerases useful?

A

Used in PCR

Taq, Vent, Pfu

26
Q

What is the Kelnow fragment of DNA Pol ONE?

A

DNA Pol without Exonuclease activity (5 to 3)

Use to synthesis oligos for radiolabelling or Chain Terminator Sequencing

27
Q

Why is T4 PN Kinase useful?

A

It transfers a P to the 5’ OH end

LABELS that end

(For fragments or oligo probes)

28
Q

What are the FOUR properties of cloning vectors? (A MUM)

A

A - Autonomous Replication

M - Marker (to select for cells)

U - Unique Restriction Sites

M - Minimal nonessential DNA
(maximise cloning size range)

29
Q

How long are PCR Primers?

A

17-25nt

30
Q

What is the temperature progression in PCR?

A

95 > 55 > 72

31
Q

What three things happen at the different temperatures in PCR?

A

Melt > Primer Anneal > Extension

32
Q

What is PCR Error Rate like?

A

Greater than DNA rep

Depends on polymerase proofreading

Low error needed for cloning

33
Q

How can you join ends cut by different REs??

A

Polymerase then Ligase

Linker ligation

Adapter ligation

Add sequences to 5’ end by PCR

34
Q

What is a Linker?

A

A DS oligo with a restriction site

35
Q

What are Adapters?

A

Pre-formed cohesive ends (blunt and sticky end)

Add by blunt ligation

36
Q

What is a cos site?

A

The sites cleaved from the thread replicated from a rolling circle, which produce cohesive ends and are packaged in the phage

37
Q

How do you make a Lambda insertion vector?

A

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
Q

What is a contig?

A

All overlapping clones formed from chromosome walking

39
Q

How do you screen PHAGE Genomic libraries?

A

96 well plating

OR

radioactive probes for gene

40
Q

How do you screen BAC genomic libraries?

A

96 well plates

Or

UCSC Genome Browser

Molecular Genetics (5 decks)

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