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Molecular Genetics > Lecture 9 - Recombinant DNA And Molecular Cloning 1 > Flashcards

Lecture 9 - Recombinant DNA And Molecular Cloning 1 Flashcards

1
Q

What does recombinant DNA engineering provide the means to?

A

Assemble genetic information in new combinations in a directed way

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2
Q

What is a key recombinant DNA technique?

A

Gene cloning

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3
Q

How does gene cloning work?

A

Gene of interest linked to a plasmid vector which enables a,privation and propagation as a pure population of molecules in a cell

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4
Q

Why is molecular cloning of DNA important? 6 reasons

A

It purifies and amplifies individual fragments of DNA of genes of interest
Helps obtain their DNA sequences
Determine the gene structure and regulation
Perform site directed mutagenesis to investigate function
Express and purify protein for biochemical/structural analysis
Enable genome analysis by creating overlapping clones of genomic DNA

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5
Q

Enzymes for recombinant DNA engineering - why is type II restriction endonucleases used for?

A

Cleave DNAs at specific sequences

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6
Q

Enzymes for recombinant DNA engineering - why is type II methylate used for?

A

Methylates dnas at specific bases

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7
Q

Enzymes for recombinant DNA engineering - why is dna polymerase used for?

A

Copy dna form a primer at 3’ end

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8
Q

Enzymes for recombinant DNA engineering - what is rna polymerase used for?

A

Makes as rna copy of dna from a promoter

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9
Q

Enzymes for recombinant DNA engineering - what is reverse transcriptase used for?

A

Makes a dna copy of rna from a primer at the 3’ end

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10
Q

Enzymes for recombinant DNA engineering - what is DNA ligand used for?

A

Covalent lay joins two dna molecules or fragments

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11
Q

Enzymes for recombinant DNA engineering - what is exonucleases used for?

A

Remove nucleotide residues from the end of dna

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12
Q

Enzymes for recombinant DNA engineering - what is terminal transferase used for?

A

Adds a homoplmer tail to the end of dna

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13
Q

Enzymes for recombinant DNA engineering - what is polynucleotide kinase used for?

A

Add a phosphate to the 3’ end of dna

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14
Q

How can strains of bacteria become resistant to bacteriophage infection?

A

The appropriate endonucleases within the bacteria recognises specific sites in infecting dna and destroys it,

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15
Q

How can strains of bacteria that are resistant to bacteriophage infection protect their own dna from being cut if it is in the same sequence as infecting bacteria?

A

Expresses cognate methylates that modify dna at corresponding sequence specific sites in dna

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16
Q

What is the requirements for type II restriction endonucleases?

A

Magnesium and not ATP

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17
Q

restriction enzymes commonly recognises what type of sequences and example?

A

Symmetric palindromic. For example BamHI recognises G cut GATCC and binds to a homodimer forming a 2-fold symmetric enzyme-DNA complex

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18
Q

Do type II restriction endonucleases cut outside their recognition sequence to one side?

A

Yes

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19
Q

How many types of termini are produced by type 2 restriction enzymes?

A

3 - 3’ recessed ends, blunt ends and 5’ recessed ends

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20
Q

What does the 5’termini of each strand retain?

A

Phosphoryl group

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21
Q

What does the 3’ termini of each product retain?

A

Hydroxylated

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22
Q

What does EcoRI produce in terms of ends?

A

Staggered end

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23
Q

What type of end does EcoRV form?

A

Blunt

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24
Q

What can block restriction enzyme cleavage of DNA?

A

Methylation added by cognate methylates

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25
Q

What does dna ligase catalyse?

A

Formation of 5 - 3 prime phosphodiester binds in ds dna or to repair strand nicks and join adjacent Okazaki fragments

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26
Q

What is the ligase cofactor?

A

RATP for t4 DNA ligase

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27
Q

In what case can DNA ligase join two fragments together?


A

Ones with comparable sticky ends with a hydroxyl group at the free 3’ end and the phosphate group at the free 5’ end

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28
Q

Can DNA ligase also join restriction fragments with blunt ends?

A

Yes

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29
Q

What do high concentrations of DNA in lifetime lead to?

A

Inter-molecular ligation producing linear DNA

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30
Q

What do low concentrations of DNA in ligase lead to?

A

Infra-molecular ligation producing circular dna

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31
Q

What can ligase do to two restriction fragments with comparable ends?

A

Link them to give a covalently closed circular product at low concentration

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32
Q

What way does DNA polymerase synthesis DNA and uses what substrate?

A

5’ to 3’ using deoxyribonuclotide triphosohate as a substrate

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33
Q

Are DNA polymerase processive?

A

Yes

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34
Q

Many DNA polymerase have a 3’ to 5’ exonuclease activity what is this and what does it do?

A

Proof reading to eliminate errors in dna synthesis

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35
Q

What do some DNA polymerase have which removes DNA ahead of them?

A

5’ to 3’ exonuclease activity

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36
Q

What does klenow sub-fragments if DNA polymerase I lack?

A

5’- 3’exonuclease activity

37
Q

What is 5’ to 3’ exonuclease activity used for?

A

Initiating DNA synthesis from oligonucleotide primers for radioactive labelling or chain terminator DNA sequencing

38
Q

Why is T4 DNA polymerase useful for trimming restrictive enzymes fragments?

A

5’ to 3’synthesis activity and high level 3’-5’ exonuclease activity

39
Q

What is used for chain terminator DNA sequences?

A

T7 DNA polymerase

40
Q

What is reverse transcriptase used to make and how does it do this?

A

Complementary DNA (cDNA) from RNA templates initiating synthesis from an oligonucleotide primer

41
Q

What thermosensitive polymerases are used for PCR?

A

Taq, Vent and Pfu

42
Q

How can DNA polymerases be used to radioactively label restriction fragments?

A

By using an alpha labelled dNTPin the polymerase reaction

43
Q

How does klenow DNA polymerse I used to make a radioactive DNA probe from the entire DNA fragment using random hexanucleotide?

A

Denature DNA and anneal hexanucleotide primers (hybridise randomly)
Synthesis of labelled dna strand
Denature dna and use probe

44
Q

What does T4 Polynucleotide Kinase do?

A

catalyzes the transfer and exchange of phosphate from the g position of ATP to the 5’hydroxyl terminus of double-and single-stranded DNA and RNA

45
Q

What is polynucleotide kinase used to label?

A

5’ end of restriction fragments or synthetic single strand oligonucleotide to use as probes using g-P32-ATP as the radioactive nucleotide; to phosphorylase PCR products made with non-phosphorylase primers ready for ligation

46
Q

Where are cloning vectors derived from?

A

Plasmid, phage or a combination of both

47
Q

How do Plasmid vectors work?

A

vector DNA introduced by transfection

48
Q

How do phage vectors work?

A

vector DNA introduced by transduction (phage infection)

49
Q

How does a combination of phage and plasmid vector work?

A

vector DNA introduced by transduction

50
Q

What are the common properties of cloning vectors?

A

Unique restriction sites to facilitate cloning of insert DNA

genetic marker(s) to select for or identify cells containing the vector

Ability to promote autonomous replication and amplify from a single copy

minimal non-essential DNA to maximize size range of cloning

51
Q

What are plasmid cloning vector features?

A

Selectable genetic marker – constitutively expressed gene encoding antibiotic drug resistance

A replication origin (ori)for autonomous replication in bacteria

• A unique restriction enzyme sites where DNA-restriction fragment

52
Q

What is the process of molecular cloning? Preparing vector DNA

A

Prepare vector DNA by cutting at the unique restriction enzyme site in plasmid to produce a linear molecule

53
Q

What is the process of molecular clone - step 2 purify?

A

Prepare and purify the DNA to be inserted by cutting with the same restriction enzyme or an enzyme that produces the same type of cohesive ends

54
Q

What is the process of molecular cloning step 3 - mixing

A

Mix dna at low concentration and add ligase

55
Q

Process of molecular cloning step 5 - transformation

A

Transformation into an E. coli strain with optimal genetic features for molecular cloning

56
Q

What is the 2 ways e.coli cells can transform with lighted DNA within?

A

(a) Using CaCl2 - treated E. coli cells and heat shock
(b) Using high voltage electroporation

57
Q

What cells survive on an antibiotic resistant place when selecting?

A

Only cells containing a plasmid survive but many may not have plasmids with an insert

58
Q

What is used to prevent self-ligation of vector ends

A

Phosphatase

59
Q

Why are modern plasmid vector designed?

A

engineered to replicate at high copy number, to be of minimal size with an artificial polylinker cloning region that contains multiple unique restriction sites, and the capability for rapid screening of recombinant s

60
Q

What does Screening for recombinant plasmids by blue/whitecolony colour test rely on?

A

E.coli strain expressing an inactive version of beta-galactosidase

61
Q

How is the activity of beta-galactosidase restored?

A

Association with an alpha peptide

62
Q

How do you screen recombinant clones by nuclei acid hybridisation?

A

Transfer onto nylon filter,

Denature dna

Hybridise to prove

Wash and expose to X-ray film

63
Q

CN ends produced by different restriction enzymes be comparable and if so give an example?

A

Yes, BamHI (5’G/GATCC3’) and BglII(5’A/GATCT3’) produce identical cohesive ends that can be efficiently ligated

64
Q

How would you legate BamH1 and Bg/II?

A

Fill in the ends or resect DNA ends of vector and insert with T4DNA polymerase plus dNTPs to make them blunt and then perform blunt end ligation

Then Fill in ends of insert and ligate linkers containing recognitions sites of vector

65
Q

How is Klenow DNA polymerase I or T4 DNA polmerase used to modify DNA-restriction fragments with recessed 3’ ends

A

Polymersiation

66
Q

How is T4 DNA polymerase used to modify DNA restriction fragments with protruding 3’ ends?

A

3’-5’ exonuclease digestion and polymerisation

67
Q

Can an EcoRI cut vector be blunt end lighted to a Sacl restriction fragment?

A

Yes

68
Q

Linker ligation strategy - what drives addition of linkers by bling end ligation making it efficient?

A

very high concentration of the duplex phosphorylated linker molecule

69
Q

Adapter ligation strategy - what is addition of adapters driven by?

A

Hugh concentration of the duplex phosphorylated linker molecule

70
Q

Genetic variants of E. coli K-12 improve the efficacy and safety of recombinant DNA cloning - How?

A

Type I restriction modification systems can potentially dstroy foreign DNA-cloned in bacterial cells

71
Q

Type I restriction modification systems can potentially dstroy foreign DNAcloned in bacterial cells - how is this done through ecoK restriction system?

A

Encodes proteins that efficiently degrade foreign DNA that is not properly methylated at the sequence 5’-AAC-(N) 5-GTGC-3‘, which would occur in many DNAs to be cloned

72
Q

Why does the hsdR- strain allow propagation of plasmids with foreign DNA?

A

the gene encoding the Type I restriction enzyme R is deletedand the plasmid is not degraded. The methylase gene M is still retained.

73
Q

What isn’t good for a successful gene cloning experiment?

A

Rearranged or deleted insert dna

74
Q

Most e.coli k12 strains used for cloning has mutations where?

A

RecA gene which encodes an essentialgene for general homologous recombination to prevent spontaneousrearrangement between repeated sequences

75
Q

How does recA enhance the bio safety of E.coli K12?

A

recAdeficient strains are sensitive to ultraviolet light

76
Q

Some mutations cause loss of the gene encoding DNA specific endonuclease. What is this called and why is this good?

A

EndA. Loss of this endonuclease greatlyincreases plasmid DNA yields and improves the quality of DNA that isisolated using standard biochemical preparations

77
Q

What does PCR obtain?

A

Pure dna in a test tube

78
Q

What is used in PCR that is stable and active in the temperature changes seen in PCR?

A

Thermostable DNA polymerase

79
Q

How does PCR work?

A

by amplification of a target DNA sequence defined by the specific hybridisation of oligonucleotide primers and their extension by DNA polymerase

80
Q

PCR needs to knowledge of target sequence at the 3’ end of PCR primers need to be complementary to what?

A

Target sequences 17-25 nucleotides long

81
Q

What is the synthesised product of copying reaction in each cycle used for?

A

a template in a subsequent reaction cycle

82
Q

What does multiple cycles of PCR leave you with?

A

massively amplified population of predominantly identical double strand DNA molecules (an amplicon) with a length size representing the distance apart of the primer pair

83
Q

PCR – the process in the initial cycles - how do you get the right length?

A

Anneal primer to DS DNA after denaturation.

Dna synthesis

Continue through many cycles until you get the correct length of DNA needed (this will occur due to the back and forth nature of this experiment)

84
Q

How do you add nucleotide sequences onto the amplicon ends using PCR?

A

Add them to the end of the PCR primer and they will be copied in throughout

85
Q

PCR can be used to amplify desired DNA with primers that contain appropriate restriction endonuclease sites in the 5’end. How does this happen?

A

Restriction site sequence in the 5’ extension is incorporated into the end of the double stranded PCR amplicon during the 2nd and 3rd round of PCR. Then amplicon is cut with appropriate restriction enzyme for each end to make cohesive ends necessary for ligation with a vector

86
Q

What does PCR error rate depend on?

A

whether or not thermostable polymerase has a proof reading function (3’ to 5’ exonuclease activity)

87
Q

What is important when considering what thermostable enzyme is required?

A

Error rate and processivity

88
Q

What is required if PCR product is to be used for molecular cloning?

A

Low error rate

89
Q

What does Long range PCR use?

A

Taq polymerase and a proof reading polymerase

Molecular Genetics (20 decks)

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