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Molecular Genetics > Recombinant DNA And Molecular Cloning 2 > Flashcards

Recombinant DNA And Molecular Cloning 2 Flashcards

1
Q

molcular cloning with lambda - can this be used as a clone? how is this done?

A

Yes and is done through using a cosmid vector

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2
Q

what appen in the phage head during infection?

A

the linear DNA is released and circulses due to the sticky ends.

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3
Q

What is the sticky end region in the circular DNA called?

A

Cos Site

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4
Q

What happens once DNA in the bacteriophage is in the bacteia?

A

It can replicate

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5
Q

What are the two pathways the phage head can go down once being released from the bacteriophage into the bacteria cell?

A

lambda lytic pathway and lysogenic pathway

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6
Q

What happens during the lysogenic pathway?

A

After circulising the DNA is integrated onto a site on the e.coli chromosome.

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7
Q

What can happen to cells in the lysogenic state?

A

they can become induced to become non-lysogenic.

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8
Q

Is the lysogenic pathway deleterious for the cell?

A

No

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9
Q

What happens in the lambda lytic pathway?

A

This happens immediatly after the DNA is released from the bacteriophage and after circulisation this undergoes feta replication to make more copies.

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10
Q

In the lambda lytic pathway what happens after the feta replication

A

These then undergo rolling circle replication which roles out the linear DNA. An enzyme called Ter (encoded by the lambda phage) then cuts at cos sites to create linear DNA which can then enter more phage heads which are made by the lambda genes.

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11
Q

lambda lytic pathway - what happens to the cell?

A

cell dies

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12
Q

What can the lysogenic cells be inducted into going down what pathway?

A

The lambda lytic pathway

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13
Q

lambda DNA packaging - one strain of e.coli with lamda in it can be induced to go into the lytic pathway, however there is an issue with this. What is that issue?

A

The lysogense have defective lambda in them - these cant make infectious phage heads. There can be issues in either protein E or protein D

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14
Q

How would you make the lysogene infectious?

A

You can make cell extracts of both of the defevt lambda and combine them before adding a lambda DNA catenane which will package invitro into phage heads.

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15
Q

converting lambda into a cloning vector - what is one issue of a lambda cloning vector?

A

Normal genomes cant contain large inserts of DNA because of size

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16
Q

How would you resolve the lambda genome size issue?

A

You would delete a region of the lambda (this would be the part which doesnt affect the lytic pathway).

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17
Q

How do you generate a lambda insertion vector?

A

cut out non essential region and ligate the DNA back together this will take around 10 kbp of sequence

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18
Q

What is an alternative way you can make a lambda replacement vector?

A

cleave all of non-essential region and substituted with a snuffer region containing restriction enzyme sites creating a replacement vector.

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19
Q

Can the stuffer region be removed to allow the DNA sequences we want in in?

A

yes

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20
Q

How exactly do we make a cloning vector - what happens to the vectro and insert DNA?

A

they are ligated together and packaged in vitro to produce infectious recombinant lambda.

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21
Q

How exactly do we make a cloning vector - what is the concentration of the ligation and what does this do?

A

high concentration making large concatemers

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22
Q

How exactly do we make a cloning vector - what happens with the arrangement of the lambda arms and what cuts them?

A

Arrangement of lambda left arm - insert- lambda right arm is correct size after cutting by Ter enzyme at cos sites,so that it can be packaged into the phage head to produce infectious particles

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23
Q

Cosmid vector cloning - what do these contain?

A

A plasmid with a phage cos site

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24
Q

How is cosmid vector cloning made?

A

the plasmid vector with the lambda cos site are ligated with fragments of new DNA.

High concentration ligation produces catenane structure

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25
Q

what happens once a cosmid vector enters into a cell?

A

They transfect not infect so cosmid plus insert circularises via cohesive ends, and is then replicated as a plasmid and is selected with ampicillin

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26
Q

How do we clone a particular gene from a complex genome?

A

Construct a genomic or complementary (cDNA) DNA library that contains a true representation of all theDNA/RNA sequences present in the starting material followed by screening the library to identify the desired clone

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27
Q

How do you amplify specific sequences from genomic DNA, or cDNA?

A

PCR using reverse transcription of mRNA.

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28
Q

How do you contruct a genomic DNA library?

A

cloning over lapping DNA fragments that have been generated by partial digestion of the genomic DNA with a restriction enzyme

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29
Q

What does cloning over lapping DNA fragments that have been generated by partial digestion of the genomic DNA with a restriction enzyme create?

A

multiple segment end points within the population of fragments

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30
Q

what enzyme is used to make partial digest?

A

depend on choice of vector – the partial digest size selection needs to be compatible with the vector insert size capacity

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31
Q

How do you use partial restriction enzyme digestion to make DNA for a genomic library?

A

Digest with Sau3A and size select through centrifugation (sucrose gradient). Direct cloning of Sau3A fragments ~ into uniqueBamHI site of a lambda replacement cloning vector.

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32
Q

How do you make a genomic library in a lambda replacement vector?

A

Lambda DNA central region of EMBL3 vector called stuffer fragment is replaced by insert produced by partial digestion (average size25 Kbp) of genome of interest

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33
Q

what will a lambda with a red/gam gene do?

A

Inhibit lytic growth

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34
Q

How do you plate a lambda library?

A

lambda library plaques are transferred onto nylon filters at high density for screening by hybridisation with a radioactive probe to gene of interest; or for plaque picking into 96 well plates for PCR screening with gene-specific primers

35
Q

what are positive plaques used to make?

A

Positive plaques are used to infect fresh cells to make more phage

36
Q

How do you make a genomic library in a cosmid vector?

A

Cell DNA prepared by partial digestion with Sau3A and ~35 Kbp size fraction ligated into BamHI site of cosmid vector

37
Q

what does each colony show?

A

a single infection from one phage head

38
Q

How do you work out the number of individual clones required in a genomic library to get close to complete covarage - what is the general idea?

A

The larger the size of DNA fragment the less clones you need to screen. The larger the size of of the genome the more you need to screen.

39
Q

what is the the next stage of getting clones: Overlapping cloned inserts enable ‘chromosome walking - how is this done?

A

Several clones are detected with probe 1 and can be aligned. Terminal regions of these clones can then be used to identify new probes (2 and 3)to screen library to identify further clones with overlapping inserts. Overlapping clones constitute a ‘contig’.

40
Q

why is chromosome walking good?

A

Faster determination of overlapping clones from multiple start points is achieved by ‘clone fingerprinting

41
Q

genomic library in a Bacterial Artificial Chromosome (BAC) vector - what are these?

A

BACs are engineered derivatives of E.coli Ffactors and replicate at 1-2copies per cell with stablemaintenance of theircloned inserts

42
Q

How do you make a genomci library in a BAC vector?

A

BAC library clones are robotically picked into 96 well plates. DNAs areprepared and then identification of a specific clone is carried out bycombinatorial PCR screening of all 96 well plates using gene specific primers

43
Q

Where can we get a BAC clone?

A

BAC library clones located in region of interest can be ordered fromGenome Resource Centres

44
Q

Making a complementary DNA (cDNA) library - how is cDNA prepared?

A

reverse transcription of mRNA

45
Q

What are cDNA libraries representitive of?

A

RNA population(cell type) from which they were derived

46
Q

How do you produce cDNA?

A

Hybridize the mRNA with a poly-T-primer and make complementary DNA copy with reverse transcriptase. You then degrade RNA with RNase H.

Then syntheisise a second cDNA strand using DNA polymerase with the RNA fragment acting as a primer.

47
Q

How do you add restriction enzyme sites using linker which improves cloning efficiency?

A
  1. Methylate the cDNA at GAATTC sites using EcoRI methylase
  2. Ligate EcoRI linkers to ends
  3. Cut with EcoRI
48
Q

how do you clone cDNA fragmetns into a lambda insertion vector?

A

single site of EcoR1 and clone the cDNA into here. This is then packaged and plated out.

the EcoR1 = clear plaques.

49
Q

How to screen cDNA library for specific clones?

A
  1. By hybridisation: DNA/RNA’ oligonucleotide probes
  2. By PCR
  3. Using antibodies to expressed proteins
  4. Functional cDNA screens in appropriate cell type
50
Q

What does Direct cloning of PCR-amplified DNA need?

A

some knowledge of the sequence and a pool of degenerate oligonucleotide primers.

51
Q

Direct cloning of PCR-amplified DNA - what is the target DNA?

A

genomic DNA or cDNA

52
Q

Direct cloning of PCR-amplified DNA - how does this work in general?

A

PCR product is cloned directly into plasmidv ectors by one of a variety of method

53
Q

Direct cloning of PCR-amplifies DNA: what is TOPO cloning and how is it done?

A

TOPO cloning uses avector with a T overhang and topoisomerase linked to ends; combine with Taq PCR product that has an extra A on each end – very efficient reaction

54
Q

Direct cloning of Odorant Receptor multi-gene family by PCR - how did they do this - what were the degenerative oligonucleotides primers?

A

conserved regions of amino acid sequence of known G protein-coupled receptors

55
Q

Direct cloning of Odorant Receptor multi-gene family by PCR - how did they do this - how did they prepare RNA from the mouse olfactory epithelium?

A

cDNA made from reverse transcription for PCR

56
Q

Direct cloning of Odorant Receptor multi-gene family by PCR - how did they do this - How many base cutters where the PCR products cut by?

A

4

57
Q

What are MULTI-COMPONENT PLASMIDS?

A

Plasmid vectors constructed with DNA sequences derived from multiple sources for numerous applications

58
Q

What are examples of multi-component plasmids and where do they come from>

A

Reporter expression vectors with fluorescent markers expressed from desired transcriptional control sequences to identify cell types expressing protein of interest by fluorescent activated cell sorting

Shuttle vectors with separate replication origins, selection markers and promoters for use in prokaryotic and eukaryotic cells

59
Q

cDNA-reprter expression plasmid vector - can they be expressed directly from promotor of interest?

A

yes

60
Q

cDNA-reporter expression plasmid vector - can they also work as part of a fusion protein or linked to a promotor cDNA expression unit via an IRIS sequence?

A

yes

61
Q

What is the point of a shuttle vector?

A

Screen

62
Q

what did a shuttle vectro for functional DNA find?

A

A nanog gene

63
Q

What does SITE-DIRECTED MUTAGENESIS OF CLONED DNA introduce?

A

single nucleotide changes, deletions or insertions of several nucleotides into cloned DNA sequence using synthesised oligonucleotides

64
Q

In what ways can you make site-directed mutagnesis of cloned DNA?

A

Change amino acid sequence of protein
Create (or remove) a restriction enzyme site
Alter gene regulatory regions to define key sequence

65
Q

What is the Site directed mutagenesis method?

A

Parent strands methylated, then denature the parent strands, anneal oligonucleotide primers (incorporate desited mutations) and add dNTPS and pfu DNA polymerase.

Extend primer and denature and reanneal. Add Dpnl

Only plasmids uncut by Dpnl survive

66
Q

What are the DNA construct assembly strategies?

A

DNA with mutliple genes can be assembled without seems in two ways: Golden gate assembly and Gibson assembly

67
Q

What does the goldon gate assembly utilise?

A

Type IIS restriction endonucleases (cleave at a distance from their recognition site) to create unique cohesive ends to allow efficient simultaneous assembly of multiple components by ligation in vitro. No restriction sites remains at ligation joining positions of final assemblys

68
Q

How does the Gibson Assembly create no joins?

A

allows DNA molecules to be joined via annealing of short end regions of complementarity in an in vitro reaction that can enable multi-segment assemblies up to 100s Kbp. No sequence ‘scar’ at joining positions of final assembly. Very flexible andfluent method to implement with few constraints on its application

69
Q

Golden gate DNA assembly - how does this work - steps?

A

Type IIs restriction enzymes such as Bsa I cleave away from their recognition site (right) at different positions on each strand

70
Q

What does cleavage during golden gate DNA assembly make?

A

This makes staggered ends with the nucleotide sequence of the extension determined by the region flanking the recognition site.

71
Q

How would you make compatable staggered ends during golden gate assembly?

A

Positioning BsaI recognition sites flanking sequences of interest that are required to bejoined can allow DNAs to be cut to create compatible staggered ends for ligation

72
Q

Can restriction enzyme digestion and ligation happen at once?

A

yes to make a one step reaction

73
Q

How can you add a Bsal site by PCR?

A

including these in the 5’ end sequences of oligonucleotide primers used for PCR

74
Q

The Gibson assembly strategy: is this a one step method?

A

Yes, and can assemble multiple dsDNA fragmetns at a time.

75
Q

Does the Gibson assembly strategy require restriction enzyme?

A

No

76
Q

How is a Gibson assembly strategy done?

A

Joining of two molecules of 4 Kbp and 3 Kbp with 450 bp overlapIn an isothermal reaction to make a circular molecule, then cut with Notl.

77
Q

Why are creating overlaps for Gibson Assembly by PCR required?

A

link regions A and B from separate plasmids and insert into vector in order: vector left end – A – B – vector right end

PCR primers each contain 3’ region of complementarity to plasmid template and 5’tail with left or right end sequence of other plasmid

78
Q

The steps of the Gibson Assembly - what does this depend on?

A

Depends on T5 exonuclease, phusion polymerase and Taq ligase.

79
Q

Steps of Gibson Assembly? What does the T5 exonuclease do?

A

T5 exonuclease degrades both strands exposing single stranded region and these regions can anneal.

80
Q

Gibson Assembly what does the phusion polymerase do?

A

The phusion polymerase extends 3’ end which catches up to the 5’ strand being degraded and this leads to ligation

81
Q

When would you use PCR in a Gibson Assembly?

A

To make overlapping nucleotides

82
Q

In Gibson assembly do you add the enzymes after PCR?

A

yes

83
Q

Do Gison Assembly involve DNA and a vector?

A

yes

Molecular Genetics (20 decks)

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