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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?

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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?

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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?

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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

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10
Q

what do phosphatase and kinase treatments do?

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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?

A
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?

A
25
Q

What is northern blotting?

A
  • used to detect specific RNA sequenced among a mixture of RNA
26
Q

What are some differenced between southern and northern blotting?

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27
Q

What are some limitations to northern blotting?

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28
Q

What is cDNA synthesis?

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29
Q

What are gene expression microarrays?

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30
Q

what do one-colour microarrays measure?

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31
Q

what are the limitations of microarrays?

A
32
Q

What is RNA-SEQ

A
  • analyses the quantity and sequences of RNA
33
Q

What is poly-A selection in RNA Seq?

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34
Q

What is a cDNA library

A
35
Q

What are the advantages of RNA Sequencing?

A
36
Q

how many genes in human DNA

A

30-40,000 genes

37
Q

What are 2 methods for altering gene expression?

A

crispr-cas9 and rnai

38
Q

Discuss the history of RNAi

A
39
Q

What are c.elans (worm)’s role in gene silencing discoveries?

A
  • dsRNA can silence genes
  • realised other key proteins are involved
40
Q

define RNAi

A
41
Q

What are the steps of RNAi?

A
42
Q

What are 2 mechanisms of silencing gene expression in RNAi?

A
  • mRNA degradation
  • translation inhibition
43
Q

What is the role of RNAi in eukaryotes?

A
44
Q

What is Ago2 (‘slicer’)?

A
  • unwinds ds siRNA
  • ## transports ss siRNA to RISC
45
Q

What are some applications of RNAi in nature and research settings?

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46
Q

What are the origins of CRISPR?

A
47
Q

What is the CRISPR-cas9 editing system?

A
48
Q

What are the 2 main components involved in CRISPR-cas9?

A
  1. guide RNA (ss)
  2. Cas9 endonuclease
49
Q

What condition must the 20bp guide sequence have to work?

A
  1. be unique
  2. lie immediately adjacent to PAM (‘5’)
    - pam is only present in the genomic DNA
    - acts as a binding signal
50
Q

What is a ribonucleoprotein?

A
51
Q

What is non-homologous end joining (NHEJ)?

A
  • pathway ideal for gene ‘loss of function’ experiments
52
Q

What is homology-directed repair (HDR)?

A
  • uses DNA repair temperature (NHEJ does not)
53
Q

What are 2 repair pathways for Crispr-induced DNA double-stranded breaks?

A
54
Q

What are some applications of CRISPR-Cas9?

A
  • study path process of diseases
  • role of genes
  • Gmo crops
  • clinical trials to correct genetically-inherited disorders

Bioc2201 (8 decks)

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