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BIOL2112 Genes and Genomes > Sequence-based cloning approaches > Flashcards

Sequence-based cloning approaches Flashcards

1
Q

What are 4 things in the traditional molecular cloning toolkit?

A

Restriction enzymes
DNA ligase
DNA polymerases
Alkaline phosphatases

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2
Q

What are restriction enzymes/restriction endonucleases?

A
  • cleave duplex DNA into fragments
  • blunt/sticky ends
  • type 2 - cleave within recognition site (4-8bp)
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3
Q

What is DNA ligase?

A
  • form recombinant DNA - joins compatible ends of duplex DNA

- from T4 bacteriophage

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4
Q

What are DNA polymerases?

A
  • Klenow fragment - lacks 5’-3’ exonuclease
  • used to fill in 5’ overhangs in presence of nucleotides
  • klenow trims 3’ overhands to form blunt ends
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5
Q

What is alkaline phosphatase?

A
  • catalyses the removal of 5’ phosphate groups

- prevents DNA ligase from making phosphodiester bond

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6
Q

What are plasmid-based vectors?

A

Natural plasmids
replicate independently of chromosome

carry antibiotic resistance gene

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7
Q

What is another term for a poly linker?

A

Multiple cloning site

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8
Q

what are the 3 stages of cloning DNA with plasmid vectors?

A

1) Generation of recombinant molecules
2) Introduction of recombinant molecules into host bacteria
3) Selection of “transform ants”

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9
Q

what are the details of stage 1 of cloning DNA with plasmid vectors (Generation of recombinant molecules)?

A

linearise with restriction enzymes

dephosphorylate with a phosphatase to prevent vector religation

ligate vector with fragment to be cloned - must have compatible ends

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10
Q

What are the details of stage 2 of cloning DNA with plasmid vectors (introduction of recombinant molecules into host bacteria)?

A

introduce DNA into host bacteria by chemical transformation (CaCl2) or electroporation

most bacteria do NOT take up plasmid - need to select!

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11
Q

What are the details of stage 3 of cloning DNA with plasmid vectors (selection of transformants)

A

Plasmid replication and bacterial cell division

spread o ampicillin plates
- cells taken up plasmid (AMPr) form colonies

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12
Q

What is the use of Xgal?

A

chromogenic substrate

hydrolyses if beta galactosidase is produced -produced insoluble blue pigment

added with IPTG for blue- white screening

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13
Q

What is IPTG?

A

analog of galactosidase - non metabolising but can induce expression of LacZdeltam15 gene in chromosome

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14
Q

What are 3 key features of the bacterial host?

A

disablement - us auxotroph

stable maintenance of transformed DNA

efficient transformation by plasmid DNA

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15
Q

What is genome annotation?

A

process of identifying the locations of genes within sequences of chromosomes - provide details of their molecular function and cellular role

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16
Q

What is the use of primers with 5’ extensions?

A

sequences at 5’ end that will not be involved in base-pairing in 1st round PCR

incorporated from 2nd round on

17
Q

Facts about Golden gate assembly?

A

single tube reaction - BsaI, DNA ligase

assembly of a vector and 1 or more DNA fragments into constructs suitable for direct transformation of a bacterial host

scarless

no recognition sites retained

18
Q

What is BsaI?

A

type IIS enzyme

non-palindromic

cuts outside recognition site

4 nucleotide overhang

can create all 256 combination

19
Q

What is the life cycle of bacteriophage lambda?

A

adopts alternative lysogenic state - genome of lambda become incorporated into genome

20
Q

What is DNA present as in phage particles?

A

linear dsDNA

single stranded terminal i of 12nt - complementary to each other

21
Q

How can nicks in E.coli be closed?

A

DNA ligase

22
Q

What is lysis in the lambda life cycle?

A

DNA replication

synthesis of phage products

particle assembly

cell lysis

23
Q

What is lysogen in the lambda life cycle?

A

integration of lambda into host genome

cells immune to further infection

24
Q

How is liquid culture assay carried out?

A

phage stock added to bacterial culture

bacterial lysis and then sometimes lysogenation occurs

25
Q

How is plaque assay carried out?

A

phage stock added to bacterial culture

aliquot spread on surface of agar plate

lysis and lysogen - turbid plaques

26
Q

How are infective phage particles formed?

A

DNA between cos sites are packaged into heads and associate with preformed tails to produce infective particles

27
Q

What is the DNA length that is packaged into a phage head?

A

78-105% of wild type lambda- 48.5kb

parts of lambda can be removed - increasing size of DNA that can be cloned

28
Q

What are lambda replacement vectors?

A

central non-essential region substituted with stuffer fragment

stuffer allows propagation in E.coli

cloning - stuffer removed and replaced by foreign insert

29
Q

Breaking the 22kb barrier or lambda vectors?

A

possible to clone larger fragments - COSMIDS

30
Q

How to make a cosmid library?

A

propagate cosmic vector as PLASMID

cleave with restriction enzyme, add foreign DNA, ligate to form concatemers, not circular products (linearise)

31
Q

How do recombinant cosmids circularise?

A

via cos sites on entry into host bacteria

32
Q

What genes to recombinant cosmids lack?

A

genes required foe lambda parcel production

33
Q

What are limitations of traditional cloning?

A

separate reactions

restricted to joining of only 2 DNA fragments, vector + insert, in single ligation

additional rounds needed to join additional fragment

can be limited by availability of suitable restriction enzyme sites

joining of large fragments problematic due to lower concentration of ends

34
Q

What are the benefits of Gibson (Isothermal assembly)?

A

assemble multiple fragments in single reaction

long fragments

scarless

any insert with appropriate overhangs can be used

35
Q

What is the process of Gibson assembly?

A

5’-exonuclease activity produces 3’ overhangs

3’ overhangs complementary base pair - produce pairs of primer and template

self-priming reaction, DNA polymerase activity extends from 3’ end of overhangs now annealed to complementary DNA to fill in the gaps

DNA ligase seals the nick - covalently links DNA fragments together

36
Q

How is it possible to join fragments of only one orientation and order by Gibson assembly?

A

use different sequences for each pair of ends to be joined

37
Q

What are some other cloning approaches?

A

Gateway recombination

TOPO

Ligation-independent

Yeast-mediated cloning & oligonucleotide stitching

Cloning by order

BIOL2112 Genes and Genomes (7 decks)

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