MolBio12-13 - 42

Question 1

Characterise restriction enzymes

Answer 1

Dimers that recognize precise palindromic DNA sequences, either cutting with overhangs, or flush (blunt)

Question 2

How do restriction enzymes work?

Answer 2

In a small volume of buffered water at 37C for 2 hours

Question 3

Where do restriction enzymes come from?

Answer 3

Identified and purified from bacteria

Question 4

Why did restriction enzymes evolve?

Answer 4

To prevent entry of foreign DNA into the cell

Question 5

How are restricted DNA fragments seperated?

Answer 5

PAGE

Question 6

Name a dye used in SDS-PAGE to stain DNA

Answer 6

Ethidium bromide

Question 7

What are cohesive termini?

Answer 7

Restricted overhangs that will bind

Question 8

Name two compatible restriction overhangs

Answer 8

XbaI and SpeI

Question 9

What is the XbaI overhang?

Answer 9

aGATC

Question 10

What is the SpeI overhang?

Answer 10

tGATC

Question 11

What are plasmid vectors?

Answer 11

Small, circular extra-chromosomal DNA that occur naturally in bacteria

Question 12

What is stereotypical of plasmid vectors?

Answer 12

Their own origin of replication - results in around 50 copies produced; usually carry antibiotic resistance genes

Question 13

How much information do plasmid vectors usually hold?

Answer 13

<30kB of DNA

Question 14

What is transformation?

Answer 14

Mixing of bacteria with plasmids, then creating temporary holes for their uptake through the membrane

Question 15

What is the problem with transformation?

Answer 15

Not very efficient

Question 16

How is the relative inefficiency of transformation overcome?

Answer 16

Antibiotic selection

Question 17

What is a cDNA library?

Answer 17

Library of DNAthat codes the transcriptome

Question 18

What is a genomic library?

Answer 18

Library of all DNA

Question 19

What is the advantage to a cDNA library?

Answer 19

Easy to compare what genes are expressed in pathological vs healthy tissue

Question 20

What is the advantage to a genomic DNA library?

Answer 20

Contains all regulatory sequences, allowing study of transcriptional regulation

Question 21

What are ESTs?

Answer 21

Expressed sequence tags

Question 22

What do ESTs do?

Answer 22

Ends all clones in the cDNA library - useful in genetic engineering

Question 23

Outline dideoxy terminator sequencing

Answer 23

Denature template, cool with primer, start DNA synthesis with DNA polymerase and tagged ddNTPs. DNA polymerase cannot extend beyond ddNTP, allowing identification of which ddNTP is at that position

Question 24

How can ddNTP sequencing be read?

Answer 24

Film (100-400 NT); automation by camera (<1000 NT)

Question 25

What is progressive sequencing?

Answer 25

Sequence from either end, restarting each time you hit about 1000 NT, until you meet in the middle

Question 26

What is shotgun sequencing?

Answer 26

Sequence loads of short random sequences (traces) and let a computer assemble them into a contig

Question 27

What is the advantage of shotgun sequencing?

Answer 27

No need for thought

Question 28

What is the disadvantage of shotgun sequencing?

Answer 28

Need to sequence more than 6x the size of the genome

Question 29

What is parallel sequencing?

Answer 29

Combination of shotgun and progressive

Question 30

When was the human genome sequenced?

Answer 30

1990-2003

Question 31

How long does it currently take to sequence a genome?

Answer 31

56 hours

Question 32

How are genes found?

Answer 32

Gene prediction software, or using a computer to translate all 6 reading frames

Question 33

How does gene prediction software function?

Answer 33

Looks for start/stop/splice sites

Question 34

Outline Blast protein alignment

Answer 34

Input a/a sequence of proposed protein, Blast searches for other proteins with similar sequences, which shows alignment of query with subject

Question 35

What does a similarity Blast result tell us?

Answer 35

Possibility that proteins evolved from same common ancestor and have similar molecular proteins

Question 36

What is a microarray?

Answer 36

Sample of 10,000s genes at once - small scale, fast and automated, each spot containg one cDNA

Question 37

In what three ways are genes ID'd?

Answer 37

cDNA library, genomic predictions, microarray interest

Question 38

How can genes be added to mice?

Answer 38

Homologous and non-homologous recombination

Question 39

What is homologous recombination?

Answer 39

Only gene with homologous arms added - nothing else

Question 40

What is non-homologous recombination?

Answer 40

Cell's DNA machinery recombines the construct (very inefficient)

Question 41

How are genes knocked out in mice?

Answer 41

Add NEO/TK complex in gene (making it not work); treat with neomycin (positive selector for NEO); treat with GANC (kills all with TK); cells introduced into mice: 1st gen = mosaic; 2nd gen = non-mosaic carriers; 3rd gen = mixture of homozygous KO and wild