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BMS109 Cell & Molecular > Genetic Engineering > Flashcards

Genetic Engineering Flashcards

1
Q

Name some types of proteins

A

Structural proteins, enzymes, signalling proteins

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2
Q

What is the function of southern blotting

A

Detect/visualise DNA in homogenate

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3
Q

Describe homogenate solutions

A

cellular/organ structure destroyed

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4
Q

Describe in situ structure

A

cellular/organ structure intact

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5
Q

How is DNA detected/visualised in situ

A

chromosomal painting/spreads

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6
Q

How is RNA detected in homogenate

A

Northern blotting

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7
Q

What does western blotting detect

A

proteins in homogenate

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8
Q

What does imunno-histochemistry detect

A

proteins in situ

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9
Q

How is RNA detected in situ

A

in situ hybridisation

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10
Q

What is a disadvantage of using homogenate

A

requires larger quantity of tissue

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11
Q

What is a disadvantage of in situ methods

A

require tissue processing

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12
Q

What is useful about in situ techniques

A

determine sub-cellular location

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13
Q

What are the techniques of blotting

A

repeated freeze/thaw, mechanical devices, detergents

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14
Q

What is the first step of the blotting technique

A

DNA is in a gel and separated by size

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15
Q

What is the second step of the blotting technique

A

transfer out of gel - blotted by electric field or capillary action

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16
Q

What is the third step of the blotting technique

A

detection with labelled probe

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17
Q

what are the factors affecting DNA migration

A

DNA size, DNA shape, Gel type, Gel concentration

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18
Q

What type of gel is best for larger fragments of DNA

A

Agarose

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19
Q

What are the main two types of gel

A

agarose or polyacrylamide

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20
Q

What does a western blot rely on

A

principle of specific antigen-antibody interaction

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21
Q

What does the western blot identify

A

presence of protein, size and relative abundance

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22
Q

What does stability of hybridisation of DNA or RNA rely on?

A

degree of match between target and probe

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23
Q

What does in situ hybridisation of RNA rely on

A

principle of hybridisation of complementary probe to target RNA

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24
Q

What can in situ detection of RNA locate

A

RNA sequences within a tissue

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25
Q

What can in situ detection of RNA detect

A

mRNA expression

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26
Q

Describe correlation between signal intensity and expression in in situ detection of RNA

A

As signal intensity increases, level of expression increases

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27
Q

What are the purposes of DNA sequencing

A

predict function of DNA sequence, identify differences, confirm sequence of engineered DNA

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28
Q

How are nucleotides linked

A

phosphodiester bond between 5’prime phosphate to 3’prime hydroxyl

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29
Q

What are nucleotide terminators tagged with

A

fluorescent label

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30
Q

What size fragments run fastest

A

small

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31
Q

What are the advantages of DNA sequencing

A

deep sequencing, whole genome in one day, RNA sequencing

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32
Q

What are the purposes of restriction enzymes in DNA sequencing

A

enable analysis of DNA, enable DNA engineering

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33
Q

What does ligase catalyse

A

formation of phosphodiester bond

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34
Q

What are the characteristics of restriction enzymes that are useful for DNA engineering

A

cut at specific sequence, produce compatible cohesive ends

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35
Q

What does PCR require

A

small amount of DNA, known starting sequence of DNA, DNA polymerase, dNTPs

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36
Q

Where are DNA primers derived from in PCR

A

start and end

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37
Q

Which DNA end is sense

A

5’ prime end

38
Q

Which DNA end is anti-sense?

A

3’ prime end

39
Q

What is the first step of PCR

A

denaturation of template DNA - 95C

40
Q

What is the second step of PCR

A

annealing of primers to template DNA - 55C

41
Q

What is the third step of PCR

A

Extension - elongation of primers till end of template - 72C

42
Q

What makes it possible for PCR to modify DNA

A

Primers are incorporated

43
Q

What are primers used for in PCR

A

add suitable restriction enzyme sites - enable DNA engineering

44
Q

what is a vector

A

DNA molecule that is maintained & replicated naturally by host organism

45
Q

What does DNA cloning involve

A

inserting piece of DNA into a host vector

46
Q

What are examples of host organisms used for DNA cloning

A

bacteria & viruses

47
Q

What are plasmids

A

circular DNA - naturally found vectors in bacteria

48
Q

What are the properties of plasmids

A

maintain and duplicate themselves

49
Q

What do plasmids contain

A

origin of replication, anti-biotic resistance gene, restriction enzymes

50
Q

What does the restriction enzyme site facilitate in plasmids

A

insertion of DNA insert

51
Q

What are the two types of plasmid?

A

Cloning plasmid, and expression plasmid

52
Q

What is the difference between cloning and expression plasmid

A

expression - more complex, has a promoter

53
Q

Where does the expression plasmid insert DNA

A

downstream of the promoter

54
Q

What are the two types of DNA library

A

genomic, cDNA libraries

55
Q

What do genomic libraries contain

A

DNA sequence of organism

56
Q

What are cDNA libraries derived from?

A

mRNA - different stages of organism

57
Q

What do cDNA libraries represent

A

expressed/transcribed part of the genome

58
Q

TRUE or FALSE - you can only have one cDNA library per organism

A

FALSE

59
Q

What are the two types of target identification

A

microarrays, yeast-2-hybrid screen

60
Q

What do RNA microarrays compare

A

transcribed genes between two tissues E.G. healthy & damaged

61
Q

What do RNA microarrays rely upon

A

principle of hybridisation between mRNAs to probes

62
Q

How many probes fo RNA microarrays need

A

enough to represent ALL known genes

63
Q

What do RNA microarrays detect

A

mRNA

64
Q

Where is the yeast-2-hybrid screen performed

A

yeast

65
Q

What do you need for a yeast-2-hybrid screen

A

protein of interest

66
Q

What does the yeast-2-hybrid screen rely on

A

principle that genes are transcribed from promoters

67
Q

What do you fish for in a yeast-2-hybrid screen

A

proteins that interact with protein of interest

68
Q

What is expression of genes dependant on

A

close proximity of transcription factor to promoter region (activation domain)

69
Q

What does fusion between POI and binding domain require

A

expression plasmid, useful yeast promoter

70
Q

What can genetic screening be used for?

A

diagnostic and predictive for mutations/disease

71
Q

What can point mutations lead to

A

abolition of restriction sites

72
Q

Why are changes in restriction sites a marker for mutation

A

they are very specific

73
Q

What are mutations in restriction sites called

A

restriction fragment length polymorphism

74
Q

Name two other techniques of target validation

A

gene knockdown using siRNA, gene knockout in transgenic mice

75
Q

what are siRNAs

A

small interfering RNAs

76
Q

What are siRNAs used for

A

reduce expression of specific genes

77
Q

Describe the term knockdown

A

partial loss of function

78
Q

What pathway does gene knockdown use

A

endogenous RNA interference pathway

79
Q

What do cells use the endogenous RNA interference pathway for

A

regulate own gene expression

80
Q

What is DICER?

A

an enzyme that cleaves free microRNA

81
Q

What does DICER produce?

A

small double stranded fragments

82
Q

What is RISC

A

RNA-induced silencing complex

83
Q

What does RISC use to get to target

A

one strand of microRNA as a guide

84
Q

What does RISC degrade

A

target mRNA

85
Q

What are the two methods of gene knockout in transgenic mice

A

pronuclear injection, gene targeting

86
Q

Where is foreign DNA injected during pro-nuclear injection

A

pro-nucleus of fertilised ova

87
Q

Which method of gene knockout creates chimeric mice

A

Gene targeting

88
Q

Where is the transgene introduced in gene targeting

A

totipotent stem cells

89
Q

What are VNTRs

A

variable number of tandem repeats

90
Q

How many VNTR variants do we have

A

2

91
Q

Which technique of gene targeting is heritable

A

Gene knockout

BMS109 Cell & Molecular (26 decks)

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