Genetic Engineering

Question 1

Name some types of proteins

Answer 1

Structural proteins, enzymes, signalling proteins

Question 2

What is the function of southern blotting

Answer 2

Detect/visualise DNA in homogenate

Question 3

Describe homogenate solutions

Answer 3

cellular/organ structure destroyed

Question 4

Describe in situ structure

Answer 4

cellular/organ structure intact

Question 5

How is DNA detected/visualised in situ

Answer 5

chromosomal painting/spreads

Question 6

How is RNA detected in homogenate

Answer 6

Northern blotting

Question 7

What does western blotting detect

Answer 7

proteins in homogenate

Question 8

What does imunno-histochemistry detect

Answer 8

proteins in situ

Question 9

How is RNA detected in situ

Answer 9

in situ hybridisation

Question 10

What is a disadvantage of using homogenate

Answer 10

requires larger quantity of tissue

Question 11

What is a disadvantage of in situ methods

Answer 11

require tissue processing

Question 12

What is useful about in situ techniques

Answer 12

determine sub-cellular location

Question 13

What are the techniques of blotting

Answer 13

repeated freeze/thaw, mechanical devices, detergents

Question 14

What is the first step of the blotting technique

Answer 14

DNA is in a gel and separated by size

Question 15

What is the second step of the blotting technique

Answer 15

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

Question 16

What is the third step of the blotting technique

Answer 16

detection with labelled probe

Question 17

what are the factors affecting DNA migration

Answer 17

DNA size, DNA shape, Gel type, Gel concentration

Question 18

What type of gel is best for larger fragments of DNA

Answer 18

Agarose

Question 19

What are the main two types of gel

Answer 19

agarose or polyacrylamide

Question 20

What does a western blot rely on

Answer 20

principle of specific antigen-antibody interaction

Question 21

What does the western blot identify

Answer 21

presence of protein, size and relative abundance

Question 22

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

Answer 22

degree of match between target and probe

Question 23

What does in situ hybridisation of RNA rely on

Answer 23

principle of hybridisation of complementary probe to target RNA

Question 24

What can in situ detection of RNA locate

Answer 24

RNA sequences within a tissue

Question 25

What can in situ detection of RNA detect

Answer 25

mRNA expression

Question 26

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

Answer 26

As signal intensity increases, level of expression increases

Question 27

What are the purposes of DNA sequencing

Answer 27

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

Question 28

How are nucleotides linked

Answer 28

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

Question 29

What are nucleotide terminators tagged with

Answer 29

fluorescent label

Question 30

What size fragments run fastest

Answer 30

small

Question 31

What are the advantages of DNA sequencing

Answer 31

deep sequencing, whole genome in one day, RNA sequencing

Question 32

What are the purposes of restriction enzymes in DNA sequencing

Answer 32

enable analysis of DNA, enable DNA engineering

Question 33

What does ligase catalyse

Answer 33

formation of phosphodiester bond

Question 34

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

Answer 34

cut at specific sequence, produce compatible cohesive ends

Question 35

What does PCR require

Answer 35

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

Question 36

Where are DNA primers derived from in PCR

Answer 36

start and end

Question 37

Which DNA end is sense

Answer 37

5' prime end

Question 38

Which DNA end is anti-sense?

Answer 38

3' prime end

Question 39

What is the first step of PCR

Answer 39

denaturation of template DNA - 95C

Question 40

What is the second step of PCR

Answer 40

annealing of primers to template DNA - 55C

Question 41

What is the third step of PCR

Answer 41

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

Question 42

What makes it possible for PCR to modify DNA

Answer 42

Primers are incorporated

Question 43

What are primers used for in PCR

Answer 43

add suitable restriction enzyme sites - enable DNA engineering

Question 44

what is a vector

Answer 44

DNA molecule that is maintained & replicated naturally by host organism

Question 45

What does DNA cloning involve

Answer 45

inserting piece of DNA into a host vector

Question 46

What are examples of host organisms used for DNA cloning

Answer 46

bacteria & viruses

Question 47

What are plasmids

Answer 47

circular DNA - naturally found vectors in bacteria

Question 48

What are the properties of plasmids

Answer 48

maintain and duplicate themselves

Question 49

What do plasmids contain

Answer 49

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

Question 50

What does the restriction enzyme site facilitate in plasmids

Answer 50

insertion of DNA insert

Question 51

What are the two types of plasmid?

Answer 51

Cloning plasmid, and expression plasmid

Question 52

What is the difference between cloning and expression plasmid

Answer 52

expression - more complex, has a promoter

Question 53

Where does the expression plasmid insert DNA

Answer 53

downstream of the promoter

Question 54

What are the two types of DNA library

Answer 54

genomic, cDNA libraries

Question 55

What do genomic libraries contain

Answer 55

DNA sequence of organism

Question 56

What are cDNA libraries derived from?

Answer 56

mRNA - different stages of organism

Question 57

What do cDNA libraries represent

Answer 57

expressed/transcribed part of the genome

Question 58

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

Answer 58

FALSE

Question 59

What are the two types of target identification

Answer 59

microarrays, yeast-2-hybrid screen

Question 60

What do RNA microarrays compare

Answer 60

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

Question 61

What do RNA microarrays rely upon

Answer 61

principle of hybridisation between mRNAs to probes

Question 62

How many probes fo RNA microarrays need

Answer 62

enough to represent ALL known genes

Question 63

What do RNA microarrays detect

Answer 63

mRNA

Question 64

Where is the yeast-2-hybrid screen performed

Answer 64

yeast

Question 65

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

Answer 65

protein of interest

Question 66

What does the yeast-2-hybrid screen rely on

Answer 66

principle that genes are transcribed from promoters

Question 67

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

Answer 67

proteins that interact with protein of interest

Question 68

What is expression of genes dependant on

Answer 68

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

Question 69

What does fusion between POI and binding domain require

Answer 69

expression plasmid, useful yeast promoter

Question 70

What can genetic screening be used for?

Answer 70

diagnostic and predictive for mutations/disease

Question 71

What can point mutations lead to

Answer 71

abolition of restriction sites

Question 72

Why are changes in restriction sites a marker for mutation

Answer 72

they are very specific

Question 73

What are mutations in restriction sites called

Answer 73

restriction fragment length polymorphism

Question 74

Name two other techniques of target validation

Answer 74

gene knockdown using siRNA, gene knockout in transgenic mice

Question 75

what are siRNAs

Answer 75

small interfering RNAs

Question 76

What are siRNAs used for

Answer 76

reduce expression of specific genes

Question 77

Describe the term knockdown

Answer 77

partial loss of function

Question 78

What pathway does gene knockdown use

Answer 78

endogenous RNA interference pathway

Question 79

What do cells use the endogenous RNA interference pathway for

Answer 79

regulate own gene expression

Question 80

What is DICER?

Answer 80

an enzyme that cleaves free microRNA

Question 81

What does DICER produce?

Answer 81

small double stranded fragments

Question 82

What is RISC

Answer 82

RNA-induced silencing complex

Question 83

What does RISC use to get to target

Answer 83

one strand of microRNA as a guide

Question 84

What does RISC degrade

Answer 84

target mRNA

Question 85

What are the two methods of gene knockout in transgenic mice

Answer 85

pronuclear injection, gene targeting

Question 86

Where is foreign DNA injected during pro-nuclear injection

Answer 86

pro-nucleus of fertilised ova

Question 87

Which method of gene knockout creates chimeric mice

Answer 87

Gene targeting

Question 88

Where is the transgene introduced in gene targeting

Answer 88

totipotent stem cells

Question 89

What are VNTRs

Answer 89

variable number of tandem repeats

Question 90

How many VNTR variants do we have

Answer 90

2

Question 91

Which technique of gene targeting is heritable

Answer 91

Gene knockout