Libraries

Question 1

What design features do primers have?

Answer 1

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

Question 2

What is the cycle of PCR?

Answer 2

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

Question 3

What alternatives can be used for PCR denaturisation?

Answer 3

alkaline denaturisation of DNA

Question 4

Which polymerases can be used for PCR?

Answer 4

Taq with no exonuclease

Kod/Pfu with 3’-5’ proof reading

Question 5

How many PCR cycles are required for production of product?

Answer 5

4

Question 6

How many PCR cycles are used for amplification?

Answer 6

24-30

Question 7

What is the outline of the cloning protocol?

Answer 7

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

Question 8

What are libraries?

Answer 8

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

Question 9

How is the genome fragmented for libraries?

Answer 9

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

Question 10

What vectors are used for library production?

Answer 10

lamda phage (20kb) 
cosmid (40kb)

Question 11

What are libraries used for?

Answer 11

PCR amplification,

functional/manipulative genomics

Question 12

How are cDNA libraries produced?

Answer 12

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

reverse transcriptase produces cDNA

Question 13

What was the original use of libraries?

Answer 13

Ordered, overlapping libraries for sequencing with radioprobes

Question 14

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

Answer 14

Shotgun approach fragments genome and sequences are aligned without ordering

Question 15

What are the types of sequencing?

Answer 15

Sanger, Illumina, Maxam Gilbert

Question 16

How is reannealing of DNA prevented during Sanger sequencing?

Answer 16

Heated to denature and high [primer]

Question 17

Which type of gene sequencing does not require librarys?

Answer 17

Illumina sequencing is continuous so doesn’t require multiple fragments

Question 18

How does Maxam-Glibert sequencing work?

Answer 18

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

Question 19

What are the 2 types of radioactivity of NTPs?

Answer 19

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

Question 20

Which Maxam-Gilbert reaction is unreliable?

Answer 20

C

Question 21

Which enzyme adds radioactive end labels to gene fragments?

Answer 21

polynucleotide kinase

Question 22

Which nucleotide structure has the highest Tm?

Answer 22

RNA:RNA hybrids

Question 23

How can libraries be screened?

Answer 23

probes to identify target fragments similar to one previously sequenced

Question 24

What factors cause high stringency probe hybridisation?

Answer 24

T close to Tm, low salt concentrations

Question 25

What affect does formamide have on Tm?

Answer 25

Lowers it by breaking H bonds

Question 26

What type of annealing do heterologous probes have?

Answer 26

Low stringency

Question 27

How can probes be labelled?

Answer 27

radioactive, antibody-enzymatic coupling or fluorescent

Question 28

What are the methods of labelling radioactive probes?

Answer 28

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

Question 29

Which end of DNA do end labels join?

Answer 29

5'

Question 30

How are DNA nicks produced?

Answer 30

Polymerase 1 5'-3' exonuclease

Question 31

How are end labels added?

Answer 31

y-32P by polynucleotide kinase

Question 32

How do riboprobes act?

Answer 32

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

Question 33

What techniques detect radioactive probes?

Answer 33

autoradiography or phosphorimager

Question 34

Which enzymes are commonly used to label probes?

Answer 34

Alkaline phosphatase, horseradish per oxidase or luciferase produce visible products

Question 35

How can probes be detected using antibodies?

Answer 35

Biotin-UTP detected by streptavidin | Digoxigenin derived UTP detected by antibodies

Question 36

What product does alkaline phosphatase produce?

Answer 36

4-nitrophenyl phosphate

Question 37

What does horseradish peroxidase produce?

Answer 37

3,3-diaminobenzidine

Question 38

What do southern blots detect?

Answer 38

DNA

Question 39

How does a southern blot work?

Answer 39

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

Question 40

What are the differences in a northern blot?

Answer 40

mRNA not DNA | Glyoxal or formaldehyde denaturisation

Question 41

Where does in situ hybridisation occur?

Answer 41

In dead, fixed tissues

Question 42

What do microarrays detect?

Answer 42

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