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GENE221 > Module Four > Flashcards

Module Four Flashcards

1
Q

what is cloning?

A

A sequence of DNA is inserted into a plasmid vector. This vector can then be put into a cell

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2
Q

what do cloned genes tell us?

A
  • the sequence of the gene and the protein
  • leads to the function of the gene
  • manipulate the gene
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3
Q

how can we manipulate the gene?

A
  • mutate the gene
  • Insert the gene into the cell or tissue of an organism
  • Make large amounts of protein
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4
Q

what is a clone?

A

a large number of identical cells or molecules with a single ancestral cell or molecule

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5
Q

what is a DNA cloning vector?

A

a carrier DNA molecule that allows attached DNA to be replicated in a cell

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6
Q

what are examples of DNA cloning vectors?

A

plasmids and viruses

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7
Q

what is a restriction enzyme?

A

an enzyme that recognizes and cleaves DNA at specific sequences

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8
Q

what is an example of a restriction enzyme?

A

EcoRI cleaves at the palindrome GGAATTC

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9
Q

what do we have to consider when cloning a gene?

A
  • What DNA do we use?
  • What vector do we use?
  • Once we have cloned our DNA what do we do?
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10
Q

why are plasmids ideal cloning vectors?

A
  • well-characterized (sequence and function of genes known)

- small and easy to purify and manipulate

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11
Q

what are the useful features of plasmid vectors?

A
  • sequence known
  • an origin of replication
  • a selectable marker
  • Unique restriction enzyme cleavage sites
  • Easy methods to screen for recombinants
  • Can often have high copy numbers
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12
Q

what must a plasmid vector contain?

A

selectable marker
an origin of replication
unique restriction enzyme site

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13
Q

what are the 4 steps for cloning a piece of DNA?

A
  • restriction
  • ligation
  • transformation
  • selection
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14
Q

what is involved in restriction?

A

the plasmid vector and the DNA fragment of interest must be digested with a restriction enzyme to produce ‘sticky ends’

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15
Q

what is involved in ligation?

A

the DNA fragment of interest must be ligated into a plasmid vector

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16
Q

what is involved in transformation?

A

the ligated vector must be transformed into a bacterial host

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17
Q

what is involved in selection?

A

bacterial colonies carrying the plasmid with the DNA fragment of interest must be selected from those that do not

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18
Q

what are the steps for making a plasmid clone?

A
  1. cut a plasmid (vector) and DNA to be cloned with the same restriction enzyme
  2. use DNA ligase to insert the DNA into the plasmid
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19
Q

what is the sequence of an EcoRI restriction site?

A

AATT

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20
Q

how does ligase join DNA?

A

through covalent bonds

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21
Q

what is hybridization?

A

when the DNA is inserted into the plasmid

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22
Q

what is the process of transformation?

A

cella are made ‘competent’ to take up DNA by treating with ice-cold CaCl2, mixed with DNA and then heat-shocked at 42 degrees celcuis

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23
Q

what is electroporation?

A

when cells are made competent to take up DNA by treatment with an electric fied

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24
Q

what are the two methods to make cells competent?

A
  • electroporation

- Treatment with ice-cold CaCl2

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25
Q

when is a bacterial colony blue on a color screening plate?

A

when the bacteria can cleave X-gal

They have active Beta-gal

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26
Q

when is a bacterial colony white on a color screening plate?

A

when the bacteria cannot cleave X-gal

Have inactive Beta-gal so the DNA insert must have disrupted the gene

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27
Q

what are the features of the pUC vector?

A
  • lacl gene encodes the repressor
  • IPTG must be added to induce the lacZ gene
  • IPTG binds the lac repressor, acts like allolactose
  • However not broken down by beta-gal
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28
Q

how does a plate with X-gal screen for vectors with the DNA insert?

A

if the vector has the DNA inserted in the gene then it will have inactive beta-gal and be unable to cleave X-gal and the colony will be white

29
Q

what are the possible products of transformation?

A 
Uncut/religated only
Insert only
Cut only
Correct with insert in vector
Empty
30
Q

When and why are pUC vectors used?

A
  • plasmid vectors used for cloning
  • Ampicillin resistancece
  • Blue/White selection
  • Need to use pUC vector with a compatible E.coli strain
31
Q

how do we express a cloned gene?

A

can use a promoter

32
Q

How can we control expression?

A

using the Ptac promoter, induced by IPTG

33
Q

how is the cloned gene product purified?

A

Pass cell lysate through a column. The tag attached to our protein that we want to isolate will bind to the column

34
Q

What is the expression vector Ptac?

A

induced by IPTG

35
Q

what is expression vector GST?

A

binds to a GHS column

36
Q

what is PreScission protease?

A

protease that can be used to easily purify protein without GST tag

37
Q

what is a luciferase assay?

A

add luciferase to a plasmid

the level of gene expression can be easily measured as the luciferase enzyme reacts with other chemicals to emit light

38
Q

what is gateway cloning?

A

does not use a specific restriction enzyme

Uses a specific recombination system used by lambda phage

The DNA insert recombines and replaces the destination vector fragment

No ligation

The clone is in an entry vector first

39
Q

how can we clone PCR products?

A

Restriction cut sites can be created on the ends of PCR products by adding to the end of the PCR primers

40
Q

What is the function of Taq DNA polymerase?

A

Adds on an adenine at #’ ends of PCR product

41
Q

What are the features of a TA cloning vector that makes it good for PCR product cloning?

A

The vector has a 3’ thymine overhang

It cannot religate on its own and so only vectors with the DNA insert will survive

42
Q

what is the max size of PCR products?

A

~10kb

43
Q

what vector is specifically designed for PCR products?

A

The TA vector

44
Q

what is Gibson assembly?

A

Can rapidly assemble multiple DNA fragments in on the reaction

45
Q

What are the requirements for Gibson assembly?

A

the DNA must have an overlapping sequence at one end

46
Q

what is the three enzyme cocktail used in Gibson assembly?

A

T5 exonuclease
Phusion polymerase
Taq ligase

47
Q

What are the features of different E.coli strains used for cloning?

A
  • Blue/white selection
  • Fast growth
  • Cloning unstable DNA
  • Specific systems
  • Large plasmids
  • preparing unmethylated DNA
48
Q

what is a genomic library?

A

A collection of all genomic DNA fragments of a given species that have been taken from one organism and inserted into a type of vector for cloning

49
Q

what is the purpose of a genomic library?

A

to have a panel of bacteria containing individual clones that represent all of the DNA in an organisms genome

50
Q

what are the useses of a genomic library?

A
  • to isolate genes for biotechnology
  • to identify the genes in an organsim
  • to obtain the genome sequence and gene function
51
Q

what are the types of genomic DNA?

A

-eukaryotic genome
-prokaryotic genomes
viral genomes

52
Q

how is DNA cut?

A

RE digest

Shear the DNA

53
Q

what are Bacterial Artificial Chromosomes?

A

Plasmids with characteristic features from the f plasmid

54
Q

what are the features of a Bacterial Artificial Chromosome?

A
  1. Low-copy number
  2. Contain genes to stabilise the plasmid
  3. Antibiotic resistance marker
  4. Can carry large DNA inserts ~75-300kb
55
Q

when were Bacterial Artificial Chromosomes developed?

A

for the Human Genome Sequencing Project

56
Q

what are the two vectors that genomic libraries can go into?

A

Plasmid

Bacterial Artificial Chromosomes

57
Q

how is a gene expression library made?

A
  • prokaryotic DNA
  • transcribed into mRNA
  • translated into proteins
  • therefore, the genomic library can be used to look for the expression of prokaryotic DNA
58
Q

what are introns?

A

gene interruptions

59
Q

what are exons?

A

expressed portion of a gene

60
Q

what is cDNA?

A

a DNA copy synthesized from mRNA using a viral enzyme, reverse transcriptase

61
Q

what is the enzyme that does reverse transcription?

A

reverse transcriptase

62
Q

how does cDNA mean that eukaryotic DNA can be inserted into Bacterial DNA?

A

The cDNA sequence has the introns removed

63
Q

what are the types of DNA libraries?

A
  • Genomic library

- Expression (cDNA) library

64
Q

what does a genomic library contain?

A

total DNA

65
Q

what does an expression (cDNA) library contain?

A

all expressed sequences. Use expression plasmid vector

66
Q

what does gene sequencing allow?

A

determine the organization of the gene

Find out what the gene encodes

Can compare the sequence with different organisms

67
Q

what are the steps in sanger sequencing?

A

strand separate the DNA to be sequenced

Anneal Primer
Extend with 4 dideoxy nucleotides, each with a different fluorescent dye

Polymerase extends primer and gets a range of extended products ended by dye-labeled terminators

68
Q

what is whole-genome shotgun sequencing?

A
  • sequence from ends of a large number of random clones
  • Assemble sequences on a computer
  • Fill in gaps with targeted sequencing later
  • It was faster and cheaper that the ordered approach
  • Automation using Sanger sequencing made this approach excellent at the time
69
Q

what are the steps of whole-genome shotgun sequencing?

A
  • sequence from ends of a large number of random clones
  • assemble sequences on a computer
  • output is a random collection of short sequences, some of the overlapping
  • overlapping reads are assembled into contigs
  • contigs are joined together

GENE221 (6 decks)

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