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Bioc2201 > Molecular Techniques > Flashcards

Molecular Techniques Flashcards

1
Q

What is DNA Cloning?

A
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2
Q

What are 2 types of vectors?

A
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3
Q

What is recombinant DNA?

A

DNA formed from 2 sources, insert and vector.

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4
Q

How are plasmids used as cloning vectors?

A
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5
Q

What are 3 general features of cloning vectors?

A
  • origin
  • polylinker
  • selectable markers
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6
Q

What is pUC19? What makes it a suitable cloning vector?

A
  • plasmic uni of California
  • has an origin or replication, allows it to be reproduced in high numbers
  • 2 selectable markers, ampicillin resistance gene and lacZa gene
  • has polylinker
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7
Q

What is the role of restriction enzymes in DNA cloning?

A
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8
Q

What are the steps of DNA cloning?

A
  • insert and vector prep
  • ligation
  • transformation
  • screening/ selection
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9
Q

discuss insert and vector preparation

A
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10
Q

what do phosphatase and kinase treatments do?

A
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11
Q

discuss ligation

A
  • add DNA ligase
  • anneals vector
  • usually use t4 DNA ligase
  • if you treat w phosphate will not ligase without insert
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12
Q

discuss transformation

A
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13
Q

what is chemical transformation

A
  • bacteria chemically treated with calcium chloride to increase the permeability of their cell membrane to incorporate DNA, producing chemically competent cells
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14
Q

what is electroprtation

A
  • creates temp pored in cell memnbrane
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15
Q

what are 2 ways of screening/ selection

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16
Q

what is antibiotic selective pressure

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17
Q

what is blue-white colony screening

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18
Q

what is insertional inactivation of the lacZa gene

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19
Q

how do you identify bacteria containing recombinant plasmids?

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20
Q

What is nucleic acid hybridisation?

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21
Q

What is DNA-DNA hybridisation?

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22
Q

What is RNA-DNA hybridisation?

23
Q

What is southern blotting?

A
  1. digest genomic DNA with RE
  2. separate it into parts. seen as a smear on agarose gel
  3. DNA is made ss and transferred from the gel to a membrane (capillary blotting). this is to make it easier to handle
  4. membrane is baked, fixing it to the blot
  5. any unhybridized probe is removed
24
Q

What are the steps in southern blotting?

25
What is northern blotting?
- used to detect specific RNA sequenced among a mixture of RNA
26
What are some differenced between southern and northern blotting?
27
What are some limitations to northern blotting?
28
What is cDNA synthesis?
29
What are gene expression microarrays?
30
what do one-colour microarrays measure?
31
what are the limitations of microarrays?
32
What is RNA-SEQ
- analyses the quantity and sequences of RNA
33
What is poly-A selection in RNA Seq?
34
What is a cDNA library
35
What are the advantages of RNA Sequencing?
36
how many genes in human DNA
30-40,000 genes
37
What are 2 methods for altering gene expression?
crispr-cas9 and rnai
38
Discuss the history of RNAi
39
What are c.elans (worm)'s role in gene silencing discoveries?
- dsRNA can silence genes - realised other key proteins are involved
40
define RNAi
41
What are the steps of RNAi?
42
What are 2 mechanisms of silencing gene expression in RNAi?
- mRNA degradation - translation inhibition
43
What is the role of RNAi in eukaryotes?
44
What is Ago2 ('slicer')?
- unwinds ds siRNA - transports ss siRNA to RISC -
45
What are some applications of RNAi in nature and research settings?
- -
46
What are the origins of CRISPR?
47
What is the CRISPR-cas9 editing system?
48
What are the 2 main components involved in CRISPR-cas9?
1. guide RNA (ss) 2. Cas9 endonuclease
49
What condition must the 20bp guide sequence have to work?
1. be unique 2. lie immediately adjacent to PAM ('5') - pam is only present in the genomic DNA - acts as a binding signal
50
What is a ribonucleoprotein?
51
What is non-homologous end joining (NHEJ)?
- pathway ideal for gene 'loss of function' experiments
52
What is homology-directed repair (HDR)?
- uses DNA repair temperature (NHEJ does not)
53
What are 2 repair pathways for Crispr-induced DNA double-stranded breaks?
54
What are some applications of CRISPR-Cas9?
- study path process of diseases - role of genes - Gmo crops - clinical trials to correct genetically-inherited disorders

Bioc2201 (8 decks)

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