Strategies for screening

Question 1

What are the main 2 screening strategies?

Non screening strategies examples?

Answer 1

Functional cloning

Positional cloning

genome sequencing, EST

Question 2

Name categories of functional cloning strategies

Answer 2

using sequence information

using information about gene expression

using function of protein

Question 3

Give the suitable screening strategy for each functional cloning instance

Transcript is super abundant in a particular source tissue

Partial nucleotide sequence known

Partial clone available

Partial polypeptide sequence known

Differential expression between two
tissues

Answer 3

Refer the table(41/58)

Question 4

How to screen a library for w/o any knowledge of NA sequence but AA sequence?

Answer 4

proteiolysis

sequence peptides

design oligonucletides

hybridize with degenerate probes or a unique guessemer with low stringency

Question 5

What are the factors to consider in oligonucleotide designing? Therefore oligonucleotides are?

Answer 5

it should hybridize to cDNA of interest only

length ~17-20nt protein - 6AA

take degeneracy into account

-synthed as pools which contain all possibe sequences with one oligo having the right seq.

Question 6

What are the disadvantage of degenerate oligo. designing?

So, variations for this method?

Answer 6

only one is correct

others can bind non specifically

-using small no of oligos,, or use single oligo

Question 7

Factors to consider when designing the least no of oligos or guessmers?

Answer 7

choose region of protein with least degenerate codons

use the knowledge of codon usage of the org..

educated guessing

Question 8

Results from experiments?l

Answer 8

lec

Question 9

Disadvantage of using guessmers?

Answer 9

the knowledge of the AA seq. of protein needs to be known has to be long than for mixed probe

Question 10

In screening a library with antibody probes,

• libraries need to be constructed in which type of vectors?
• cDNA position to be?
• product is produced as a?

-this library can be screened with?

Answer 10

• in expression vectors
• adjacent to a promoter in correct orientation
• as a fusion protein
• antibody(if available) or raise antibodies to previously purified protein

Question 11

Give an example vector?

How to screen a library with antibody probes? (method)

Answer 11

lambda gt 11

phages are mixed with bacteria

plated out

plaques transferred to NC filter

incubated with a specific antibody to screen(Iry Ab)

IIry Ab is added which bind to Iry Ab (IIry Ab carries a label - 125I or alk.phosphotase)

expose to film and identify signals

pick corresponding plaque, purify and amplify

Question 12

sequence analysis using bioinformatics

• steps?
• how they are analyzed?

Answer 12

slides

Question 13

For what seq. data can be used for?

Answer 13

to identify coding regions, reg elements and mutations

organization of genes

resulting protein and their structure, properties and likely function

find similarities

identification, determine possible function