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Y2S2 - Adv. Molecular Biology > MolBio12-13 - 42 > Flashcards

MolBio12-13 - 42 Flashcards

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1
Q

Characterise restriction enzymes

A

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

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2
Q

How do restriction enzymes work?

A

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

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3
Q

Where do restriction enzymes come from?

A

Identified and purified from bacteria

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4
Q

Why did restriction enzymes evolve?

A

To prevent entry of foreign DNA into the cell

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5
Q

How are restricted DNA fragments seperated?

A

PAGE

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6
Q

Name a dye used in SDS-PAGE to stain DNA

A

Ethidium bromide

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7
Q

What are cohesive termini?

A

Restricted overhangs that will bind

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8
Q

Name two compatible restriction overhangs

A

XbaI and SpeI

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9
Q

What is the XbaI overhang?

A

aGATC

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10
Q

What is the SpeI overhang?

A

tGATC

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11
Q

What are plasmid vectors?

A

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

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12
Q

What is stereotypical of plasmid vectors?

A

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

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13
Q

How much information do plasmid vectors usually hold?

A

<30kB of DNA

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14
Q

What is transformation?

A

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

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15
Q

What is the problem with transformation?

A

Not very efficient

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16
Q

How is the relative inefficiency of transformation overcome?

A

Antibiotic selection

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17
Q

What is a cDNA library?

A

Library of DNAthat codes the transcriptome

18
Q

What is a genomic library?

A

Library of all DNA

19
Q

What is the advantage to a cDNA library?

A

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

20
Q

What is the advantage to a genomic DNA library?

A

Contains all regulatory sequences, allowing study of transcriptional regulation

21
Q

What are ESTs?

A

Expressed sequence tags

22
Q

What do ESTs do?

A

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

23
Q

Outline dideoxy terminator sequencing

A

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

24
Q

How can ddNTP sequencing be read?

A

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

25
Q

What is progressive sequencing?

A

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

26
Q

What is shotgun sequencing?

A

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

27
Q

What is the advantage of shotgun sequencing?

A

No need for thought

28
Q

What is the disadvantage of shotgun sequencing?

A

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

29
Q

What is parallel sequencing?

A

Combination of shotgun and progressive

30
Q

When was the human genome sequenced?

A

1990-2003

31
Q

How long does it currently take to sequence a genome?

A

56 hours

32
Q

How are genes found?

A

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

33
Q

How does gene prediction software function?

A

Looks for start/stop/splice sites

34
Q

Outline Blast protein alignment

A

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

35
Q

What does a similarity Blast result tell us?

A

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

36
Q

What is a microarray?

A

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

37
Q

In what three ways are genes ID’d?

A

cDNA library, genomic predictions, microarray interest

38
Q

How can genes be added to mice?

A

Homologous and non-homologous recombination

39
Q

What is homologous recombination?

A

Only gene with homologous arms added - nothing else

40
Q

What is non-homologous recombination?

A

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

41
Q

How are genes knocked out in mice?

A

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

Y2S2 - Adv. Molecular Biology (14 decks)

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