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Molecular Genetics 3 > 9.+10. Recombinant DNA engineering > Flashcards

9.+10. Recombinant DNA engineering Flashcards

1
Q

Explain what are restriction endonucleases

A

Restriction endonucleases: cut DNA at recognition sites in the middle of sequence (not the ends)

  • bind to DNA non-specifically - slide until recognition site found - bind specifically
  • often symmetric (palindromic) restriction sites (ex G/GAATCC)
  • Mg2+ cofactor
  • leaves cut termini: 3’ staggered / blunt / 5’ staggered
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2
Q

What are the enzymes used in recombinant DNA engineering?

A
  • Type II restriction endonucleases
  • Type II methylases
  • DNA polymerase
  • RNA polymerase
  • Reverse transcriptase
  • DNA ligase
  • Exonuclease
  • Terminal transferase
  • Polynucleotide kinase
  • Alkaline phosphotase

endo-exo
DNA-RNA pol
rev trans
lig-kin-transf
meth-ph

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3
Q

Explain what are restriction methylases

A

Restriction methylases: methylate restriction sites to prevent cutting by restriction endonucleases

-methylation added by:
- cognate methylase
- other DNA methylation patterns

BUT: DpnI cuts also methylated DNA restriction sites

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4
Q

Explain what are DNA polymerases

A

DNA polymerases: copy DNA from primer 3’ end

  • synthesis 5’ -> 3’
  • uses dNTPs
  • DNA polymerases processive - catalyze multiple polymerisations
  • 3’ to 5’ exonuclease activity - for proofreading (some also 5’ to 3’ exonucleases to remove DNA ahead)
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5
Q

Explain what are RNA polymerases

A

RNA polymerases: copy DNA sequence into RNA starting at promoter (transcription)

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6
Q

Explain what are reverse transcriptases

A

Reverse transcriptase: make DNA copy from RNA starting at 3’ primer

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7
Q

Explain what are DNA ligases

A

DNA ligases: joins two DNA backbone strands
- catalyse formation 5’-3’ phosphodiester bond (PO4 5’ + OH 3’)
- in DNA replication joins Okazaki fragments
- joins compatible sticky/ blunt ends
- T4 DNA ligase - rATP cofactor (forms intermediate with enzyme)

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8
Q

Explain what are exonucleases

A

Exonucleases: remove nucleotide residues from DNA ends

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9
Q

Explain what are terminal transferases

A

Terminal transferases: add homopolymer tails to DNA ends

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10
Q

Explain what are polynucleotide kinases

A

Polynucleotide kinases: add Pi from ATP to 5’ of DNA / RNA

  • polynucleotide kinase: radioactively label 5’ of restriction fragments / ss oligonuclotides to use as probes
  • T4 polynucleotide kinase: catalyses exchange of gamma position Pi from ATP to ss/ds DNA/RNA 5’ end (OH terminus)
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11
Q

Explain what are alkaline phosphotases

A

Alkaline phoshotases: remove terminal Pi from DNA ends

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12
Q

How are restriction endonucleases used as protection?

A

Some bacteria use restriction endonucleases as protection against bacteriophages - enzymes cleave DNA - destroy - no insertion

Bacteria protect own genome from those restriction endonucleases by methylating recognition sites - cognate methylases add methyl group - no cutting

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13
Q

What are the types of termini that restriction endonucleases could leave?

A

Cut termini could be:
- 3’ staggered
- Blunt
- 5’ staggered

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14
Q

What can prevent restriction endonucleases from cutting DNA?

A

Recognition site methylation prevents restriction endonuclease from cutting - methylation by methylases (cognate methylase)

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15
Q

How can ligation be manipulated to produce intra-molecular or inter-molecular ligation?

A

DNA ligase can join ends of different molecules / same:
- intra-molecular: add low DNA conc - joins itself -> circular DNA
- inter-molecular: add high DNA conc - joins diff molecules -> linear DNA

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16
Q

What type of ligation performed when two restriction fragments are linked into circular DNA molecule?

A

Both intra and inter-molecular ligation performed:
1) Inter-molecular joins one end of two linear molecules => linear
2) Intra-molecular joins two ends of one linear molecule joined => circular

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17
Q

Different types of DNA polymerases bbz ar reik nesamone

A
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18
Q

How can DNA polymerase be used to study restriction fragments or create detectable probes?

A
  • DNA polymerase can add radioactively labelled alpha-dNTPs
  • DNA polymerase can make radioactive DNA probe from hexanucleotide primers
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19
Q

What is a homopolymer tail?

A

Homopolymer tail: sequence added to blunt-ended polymers to join a ds DNA fragment into a cloning vehicle

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20
Q

What are the possible cloning vector sources for use in E. coli?

A

Vector sources:
- Plasmid derived - transfection
- Phage derived - transduction
- Combination of plasmid + phage - transduction

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21
Q

What are the common cloning vector properties?

A

Common vector properties:
- can promote autonomous replication
- can amplify from single copy
- have a genetic marker to select for / identify cells with vector (ex antibiotic resistance)
- restriction sites for inserting
- have minimal non-essential DNA

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22
Q

What are the examples of currently available cloning vectors?

A
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23
Q

Explain the process of molecular cloning

A

Process of molecular cloning:
1) Restriction enzymes for vector
2) Restriction enzymes for purified DNA - same type of termini (5’/3’/blunt)
3) Mix ligase + linear vector + target DNA at low conc (for inter-molecular ligation) -> will get vector+DNA / self-ligated vector
4) Transformation into E. coli

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24
Q

Explain the process of E. coli transformation with molecular vector

A

E. coli tranformation with vector:
1) Transform E. coli with vector by:
- CaCl2 + heat shock
- high voltage electroporation
2) Plate on + antibiotic plate
3) Only transformed E. coli will grow - have antibiotic resistance gene in plasmid

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25
What can be used to prevent vector self-ligation in molecular cloning?
**Phosphatases** can be used to prevent self-ligation in vectors: - **exchange 5' OH into Pi** - but vector and insert DNA can still ligate - **only one Pi in the way** - once one strand ligated - other can be ligated by E. coli repair system
26
Explain the process of recombinant clone screening by nucleic acid hybridization
27
Explain screening of recombinant plasmids using blue/white clony test
Blue/white: **inactive** version of **beta-galactosidase** => white **alpha-peptide** restores **activity** -> blue
28
How can vector and DNA insert be ligated if they cut with different restriction endonucleases?
Diff restriction endonuclease cut ends can be compatible: if cut same by both endonucleases; if **ends are modified**: - use **T4 DNA polymerase** + **dNTPs** -> make blunt -> blunt end ligation OR - add **DNA end inserts** - **ligate linkers** with vector recognition site Ex: EcoRI cut end can be ligated to SacI cut end
29
Explain how ends can be extended / shortened
DNA cut with restriction endonucleases can be modified to get needed sticky/blunt ends: - **extended**: **Klenow** DNA pol I / **T4** **DNA pol** for recessed **3'** ends - **shortened**: **T4** DNA polymerase has **exonuclease** activity - digestion of **3'** protruding ends
30
Explain how ends can be ligated using a linker?
**Linker ligation** strategy (**blunt -> sticky** ends): - **ligate multiple linkers** - **short blunt sequences added** to ends of cut DNA fragment - use restriction **endonuclease** to **get needed ends**
31
Explain how ends can be ligated using adapter ligation strategy
**Adapter ligation** strategy (**blunt -> sticky** ends): - ligate adaptors - **short DNA sticky sequences** - add polynucleotide **kinase** to **add Pi to ends**
32
What are K-12 E. coli strains?
K-12 E. coli - modified strain that **doesn't colonise human intestine** - **lacks O antigen** - **EcoK** **restriction** system **degrades** **foreign DNA** that is **not methylated** at specific sequence - only **hsdR- strain** allows **plasmid expression** - **restriction** **enzyme R** is **deleted** -> plasmid not degraded (even though not methylated)
33
Smth more on K-12 E. coli
34
Explain the process of PCR
35
Explain what is a λ bacteriophage
λ bacteriophage: - **infects E. coli** - used as a vector - **linear DNA** - **cos sites** at both ends - **cohesive ends** - can circularise - **lytic + lysogenic** pathways
36
What is λ bacteriophage DNA like?
λ bacteriophage DNA; - **linear** - canc circularise - **cos sites**
37
Explain lytic and lysogenic λ bacteriphage cycles
**Lytic**: - infects bacterium - **rolling circle replication** - mutiple copies - capsid proteins produced -> **new bacteriophage** assembly -> **burst** **Lysogenic**: - infects bacterium - **integrates into chromosome** -> **replicates**
38
Explain rolling circle replication
Rolling circle replication (RCR): -
39
Explain rolling circle replication in λ bacteriphages
40
Explain the process of λ DNA packing into bacteriophages in vitro
λ bacteriophage packaging in vitro: 1) E. coli **lysed** - contents out 2) **Ligated λ DNA** catenanes are **cut** in vitro by **Ter at cos sites** - genes for packaging 3) From lysates **proteins assemble** + **package λ DNA** -> **bacteriophages assembled**
41
How can λ be converted into cloning vector?
λ can be **converted into cloning vector**: 1) Can't simply insert into λ genome - will be **too large** to fit inside phage head -> **cut out non-essential** region 2) Inserts possible - **insert** stuffer region (**restriction sites)** - vector made 3) λ phage assembly in vitro => infections **recombinant λ phages**
42
What is the λ genome map?
λ genome: - **essential region**: capsid components, early/late regulation, DNA synthesis, host lysis - **non-essential region**: integration and excision
43
What is a replacement vector?
Replacement vector: **λ DNA cloning vector** produced by **cutting** out **non-essential regions** and **inserting cloning genes** - possible because of λ phage packaging mechanism
44
λ vetor making pic phage assembly in vitro
45
What is a cosmid vector?
Cosmid vector - **plasmid with λ cos site ligated** with new **~35Kbp DNA inserts** **High conc** ligation produced **catenane structure** for **in vitro packaging** Efficient in bacteria infection - in cells **cosmid vector** + **DNA insert circularise** via **cos** - replicated as plasmid (Amp+)
46
What are the differences between λ vector and cosmid vector?
**λ vector**: - **bacteriophage** **Cosmid** vector: - **designed plasmids** - bacterial oriV - antibiotic selection marker - cloning site - **cos sites** from λ bacteriophage
47
What is λ phage catenane structure?
??
48
How can a gene from a complex organism be cloned?
Cloning a gene frmo complex organism: 1) **Construct** **genomic** / **cDNA library** (contains representation of all DNA/RNA in starting material - screening the library to identify desried clone) 2) **PCR** to aplify specific sequence in genomic DNA/ cDNA (reverse transcription) 3) Transfect bacteria
49
What is the difference between genomic DNA libraries and cDNA libraries?
**Genomic** library: **all genes** of an organism **cDNA** library: **genes expressed** by a **specific cell **type in an organism (constructed from expressed mRNAs - reverse transcriptase-> DNA)
50
How is a genomic library constructed?
Genomic library is constructed **cloning overlapping DNA fragments** generated by **partial digestion** of genomic DNA with restriction enzymes (cut to be compatible with vector)
51
Why DNA fragments are overlapping in genomic DNA libraries?
In genomic DNA libraries DNA fragments overlap because xxx??
52
How is a genomic library made in a λ replacement vector?
53
Explain λ library plating
54
How is a genomic library made in a cosmid vector?
55
How to calculate the number of individual clones required in a genomic library to approach complete coverage?
56
Explain what is chromosome walking
Chromosome walking: **hybridization of one library clone to another** can **find overlapping** regions of DNA, and hence can be **used for identifying upstream** and **downstream** sequences from the marker One probe binds - polymerization - site for another probe
57
What is a BAC vector?
Bacterial artificial chromosome (BAC): an **engineered derivative of E. coli F factor** - replicate **1, 2 copies** per cell - stable maintenance for their DNA inserts - best for **genomic lib construction** with inserts of 200-300 Kbp - once insert is ligated into BAC - electorporated into E. coli
58
Explain BAC colony plating
59
Explain the process of cDNA production
1) **mRNA** purified 2) **Oligo-dT** tail added (**complimentary** to **polyA** tail) 3) **Reverse transcriptase** used to make complementary DNA to RNA 4) RNA degraded **RNase** 5) **DNA pol** used to **synthesise** second complementary DNA strand
60
How can cloning efficiency be improved?
1) **Methylate** DNA 2) **Add** **restriction** enzyme **sites** using linkers 3) **Cut** with restriction enzymes => **better ends** for ligation into vector
61
How can cDNA fragments be cloned into λ insertion vectors?
?? on cDNA mentioned
62
How can a cDNA library be screened for specific clones?
Ways in which cDNA libraries can be screened for clones: - **hybridization** - DNA/RNA oligo nucleotide probes - **PCR** - Using **Ab** **for expressed protein** - **functional cDNA screens** in specific cell types
63
Explain cloning PCR amplified DNA
64
How can gene clones be used in research?
To **identify genes** using related species amplified genes as **probes**
65
What is a multi-component plasmid?
Multi-component plasmid: **plasmid** with **inserts** form **different sources**
66
How can expression of recombinant DNA be visualised?
**Reporter** expression - reporter **incorporated into vector** along with inserted fragment - reporter genes - ex GFP - reporter genes can be expressed: 1) directly **from promoter** 2) as part of a **fusion protein** 3) linked to **promoter-cDNA expression unit** (via IRES)
67
What is a shuttle vector?
Shuttle vector: vector that can **propagate in two different host species** - inserted DNA can be tested in two different cell types
68
Explain functional cDNA screening for a particular gene
cDNA screening: **screening** libraries of **cDNA clones** rely on the selective **binding** of **nucleic acid probes** to cDNA / **Ab** to gene protein product encoded by the cDNA
69
What is site-directed mutagenesis of cloned DNA?
Site-directed mutagenesis: **specific nucleotide changes** (deletions or insertions) of single / several nucleotides **in cloned DNA**
70
What could be the outcomes of site-directed mutagenesis of cloned DNA?
Site-directed mutagenesis of cloned DNA could result in: - **changed am a** sequence - a **restriction site** - **altered** **gene regulatory** regions
71
What are the two DNA construct assembly strategies?
DNA construct assembly strategies: - **Golden Gate assembly**: **Type IIS** restriction endonucleases - **ligation** in vitro - **Gibson assembly**: DNA joined via **annealing** in vitro
72
Explain 'Golden Gate' assembly strategy
'Golden Gate' assembly: 1) **Type IIS restriction enzyme** cleaves away from recognition site - leaves **staggered** ends 2) Position **flanking sequences** - DNA can be **cut** to create **compatible staggered ends** 3) Ligation
73
How can restriction sites for 'Golden Gate' assembly be introduced into vector?
**BsaI sites** introduced by incorporating them into **oligo PCR primers**
74
Overview of 'Gibson' assembly strategy
'Gibson' assembly: **independent seamless joining** - ends edited - joined
75
Explain 'Gibson' assembly strategy
76
What was 'Gibson' assembly originally created for?
'Gibson' assembly was create to **generate synthetic genomes**
77
Lecture overview

Molecular Genetics 3 (17 decks)

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