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Genetics > Libraries > Flashcards

Libraries Flashcards

1
Q

What design features do primers have?

A 
Tm ~55'C
3' G/C
No long complementary repeats
20-30nt long
50% G/C
Start/stop codon
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2
Q

What is the cycle of PCR?

A

94’C denaturisation
55’C annealing
72’C extension

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3
Q

What alternatives can be used for PCR denaturisation?

A

alkaline denaturisation of DNA

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4
Q

Which polymerases can be used for PCR?

A

Taq with no exonuclease

Kod/Pfu with 3’-5’ proof reading

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5
Q

How many PCR cycles are required for production of product?

A

4

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6
Q

How many PCR cycles are used for amplification?

A

24-30

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7
Q

What is the outline of the cloning protocol?

A

Purify vector and gene fragment from library
Isolate gene by restriction digestion of PCR
Ligate gene and vector
Transform into colonies
Check recombination by antibiotic resistance or PCR
Extract vector
Transform into expression colony
Isolate protein product

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8
Q

What are libraries?

A

Collection of fragments of the entire genome contained within a collection of colonies

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9
Q

How is the genome fragmented for libraries?

A

Partial digestion by high frequency restriction sites (Sau every 250bp) or mechanical

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10
Q

What vectors are used for library production?

A 
lamda phage (20kb) 
cosmid (40kb)
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11
Q

What are libraries used for?

A

PCR amplification,

functional/manipulative genomics

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12
Q

How are cDNA libraries produced?

A

mRNA isolated by oligo(dT) columns for Poly(A) tail

reverse transcriptase produces cDNA

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13
Q

What was the original use of libraries?

A

Ordered, overlapping libraries for sequencing with radioprobes

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14
Q

What is the modern approach to sequencing of gene fragments from libraries?

A

Shotgun approach fragments genome and sequences are aligned without ordering

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15
Q

What are the types of sequencing?

A

Sanger, Illumina, Maxam Gilbert

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16
Q

How is reannealing of DNA prevented during Sanger sequencing?

A

Heated to denature and high [primer]

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17
Q

Which type of gene sequencing does not require librarys?

A

Illumina sequencing is continuous so doesn’t require multiple fragments

18
Q

How does Maxam-Glibert sequencing work?

A

4 reactions of chemical cleavage: A>G, G>A, C+T, C. Strands radioactively labelled and separated by electrophoresis for sequencing

19
Q

What are the 2 types of radioactivity of NTPs?

A

alpha at first phosphate incorporated
beta
gamma at last phosphate released as PPi

20
Q

Which Maxam-Gilbert reaction is unreliable?

A

C

21
Q

Which enzyme adds radioactive end labels to gene fragments?

A

polynucleotide kinase

22
Q

Which nucleotide structure has the highest Tm?

A

RNA:RNA hybrids

23
Q

How can libraries be screened?

A

probes to identify target fragments similar to one previously sequenced

24
Q

What factors cause high stringency probe hybridisation?

A

T close to Tm, low salt concentrations

25
Q

What affect does formamide have on Tm?

A

Lowers it by breaking H bonds

26
Q

What type of annealing do heterologous probes have?

A

Low stringency

27
Q

How can probes be labelled?

A

radioactive, antibody-enzymatic coupling or fluorescent

28
Q

What are the methods of labelling radioactive probes?

A

Single or multiple end labelling, nick translation, random hexamer extension, riboprobes, PCR

29
Q

Which end of DNA do end labels join?

A

5’

30
Q

How are DNA nicks produced?

A

Polymerase 1 5’-3’ exonuclease

31
Q

How are end labels added?

A

y-32P by polynucleotide kinase

32
Q

How do riboprobes act?

A

incorporated into a vector under control of a phage polymerase promoter
produce RNA:RNA hybrids that are resistant to ribonucleases

33
Q

What techniques detect radioactive probes?

A

autoradiography or phosphorimager

34
Q

Which enzymes are commonly used to label probes?

A

Alkaline phosphatase, horseradish per oxidase or luciferase produce visible products

35
Q

How can probes be detected using antibodies?

A

Biotin-UTP detected by streptavidin

Digoxigenin derived UTP detected by antibodies

36
Q

What product does alkaline phosphatase produce?

A

4-nitrophenyl phosphate

37
Q

What does horseradish peroxidase produce?

A

3,3-diaminobenzidine

38
Q

What do southern blots detect?

A

DNA

39
Q

How does a southern blot work?

A 
Electrophoresis 
Acid degradation of large fragments 
Alkali denaturisation 
Fixation to nitrocellulose by UV or heat
Non specific prehybridisation 
Probe hybridisation and detection
40
Q

What are the differences in a northern blot?

A

mRNA not DNA

Glyoxal or formaldehyde denaturisation

41
Q

Where does in situ hybridisation occur?

A

In dead, fixed tissues

42
Q

What do microarrays detect?

A

mRNA or cDNA annealing to immobilised DNA, multiple genes at any time

Genetics (4 decks)

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