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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
when is a bacterial colony blue on a color screening plate?
when the bacteria can cleave X-gal | They have active Beta-gal
26
when is a bacterial colony white on a color screening plate?
when the bacteria cannot cleave X-gal | Have inactive Beta-gal so the DNA insert must have disrupted the gene
27
what are the features of the pUC vector?
- 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
28
how does a plate with X-gal screen for vectors with the DNA insert?
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
what are the possible products of transformation?
``` Uncut/religated only Insert only Cut only Correct with insert in vector Empty ```
30
When and why are pUC vectors used?
- plasmid vectors used for cloning - Ampicillin resistancece - Blue/White selection - Need to use pUC vector with a compatible E.coli strain
31
how do we express a cloned gene?
can use a promoter
32
How can we control expression?
using the Ptac promoter, induced by IPTG
33
how is the cloned gene product purified?
Pass cell lysate through a column. The tag attached to our protein that we want to isolate will bind to the column
34
What is the expression vector Ptac?
induced by IPTG
35
what is expression vector GST?
binds to a GHS column
36
what is PreScission protease?
protease that can be used to easily purify protein without GST tag
37
what is a luciferase assay?
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
what is gateway cloning?
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
how can we clone PCR products?
Restriction cut sites can be created on the ends of PCR products by adding to the end of the PCR primers
40
What is the function of Taq DNA polymerase?
Adds on an adenine at #' ends of PCR product
41
What are the features of a TA cloning vector that makes it good for PCR product cloning?
The vector has a 3' thymine overhang | It cannot religate on its own and so only vectors with the DNA insert will survive
42
what is the max size of PCR products?
~10kb
43
what vector is specifically designed for PCR products?
The TA vector
44
what is Gibson assembly?
Can rapidly assemble multiple DNA fragments in on the reaction
45
What are the requirements for Gibson assembly?
the DNA must have an overlapping sequence at one end
46
what is the three enzyme cocktail used in Gibson assembly?
T5 exonuclease Phusion polymerase Taq ligase
47
What are the features of different E.coli strains used for cloning?
- Blue/white selection - Fast growth - Cloning unstable DNA - Specific systems - Large plasmids - preparing unmethylated DNA
48
what is a genomic library?
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
what is the purpose of a genomic library?
to have a panel of bacteria containing individual clones that represent all of the DNA in an organisms genome
50
what are the useses of a genomic library?
- to isolate genes for biotechnology - to identify the genes in an organsim - to obtain the genome sequence and gene function
51
what are the types of genomic DNA?
-eukaryotic genome -prokaryotic genomes viral genomes
52
how is DNA cut?
RE digest | Shear the DNA
53
what are Bacterial Artificial Chromosomes?
Plasmids with characteristic features from the f plasmid
54
what are the features of a Bacterial Artificial Chromosome?
1. Low-copy number 2. Contain genes to stabilise the plasmid 3. Antibiotic resistance marker 4. Can carry large DNA inserts ~75-300kb
55
when were Bacterial Artificial Chromosomes developed?
for the Human Genome Sequencing Project
56
what are the two vectors that genomic libraries can go into?
Plasmid | Bacterial Artificial Chromosomes
57
how is a gene expression library made?
- prokaryotic DNA - transcribed into mRNA - translated into proteins - therefore, the genomic library can be used to look for the expression of prokaryotic DNA
58
what are introns?
gene interruptions
59
what are exons?
expressed portion of a gene
60
what is cDNA?
a DNA copy synthesized from mRNA using a viral enzyme, reverse transcriptase
61
what is the enzyme that does reverse transcription?
reverse transcriptase
62
how does cDNA mean that eukaryotic DNA can be inserted into Bacterial DNA?
The cDNA sequence has the introns removed
63
what are the types of DNA libraries?
- Genomic library | - Expression (cDNA) library
64
what does a genomic library contain?
total DNA
65
what does an expression (cDNA) library contain?
all expressed sequences. Use expression plasmid vector
66
what does gene sequencing allow?
determine the organization of the gene Find out what the gene encodes Can compare the sequence with different organisms
67
what are the steps in sanger sequencing?
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
what is whole-genome shotgun sequencing?
- 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
what are the steps of whole-genome shotgun sequencing?
- 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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