Strategies for gene isolation

Question 1

What is done in DNA cloning?

Answer 1

amplification of donor DNA using a suitable replicon

Question 2

Give examples for types of cloning vectors?

Answer 2

plasmids

viruses

chromosomes

• BACS
• PACS
• YACS

others

• cosmids - plasmids containing cos sites thus can be packaged into capsids
• phagemids - plasmids containing bacteriophage m13 ori of repli.

Question 3

What are the properties of an ideal vector?

Answer 3

episomal - dont integrate into the host genome

replicates autonomously

marker

versatile - unique restriction sites, ancillary functions

high copy number

Question 4

What are the 3 major steps involved in cloning?

Answer 4

Ligate donor dna to the vector

transfer recombinant dna to the cells

plate out cells

isolate cloned dna

Question 5

What are the 2 types of DNA libraries?

Answer 5

genomic

cDNA - from mRNA by reverse transcriptase

Question 6

How to isolate a gene?

Answer 6

construct a library and screen for it

Question 7

What is a genomic library?

Answer 7

a collection of recombinant clones that together contain all the DNA present in an organism.

Question 8

Properties of an ideal genome library?

Answer 8

should have sequences that represent the entire genome

should be easy to construct from small amounts

Insert sizes - large enough that it can contain whole genes with flanking, not too large that it makes it harder to do gene mapping using rest. enz.

should be easy to isolate cloned sequence

screening should be easy

should be able to amplify library w/o loss or misrepresentation

should be able to store for long periodss

Question 9

In general what is the size of a library of a complex eukaryotic genome

Answer 9

10^5 - 10^6

Question 10

lambda phage characteristics

Answer 10

size - 48.6kb

~40% not essential for propagation of phage

Question 11

Types of lambda phage vectors? explain each.

Answer 11

Insertional ~ 12kb

substitutional ~ 23kb

Question 12

Why can’t just naked DNA be inserted into bacteria? Solution?

Answer 12

transfection

efficiency is low(okay for small genomes)

phage DNA is packaged into capsids before introduction into bacteria

Question 13

On what the no. of recombinants required to make a genomic library depends on?

Answer 13

genome size of the organism

size of cloned fragments

Question 14

Note the equation used to calculate the no. of clones required

Answer 14

N = ln(1-p)/ln(1-x/y)

Question 15

Properties of donor DNA to be cloned and what to look for?

Answer 15

DNA should be high molecular weight

shearing of dna should be avoided

• mechanical shearing
• degradative enzymes

Thus isolation procedures should have minimum no. of steps

Question 16

Explain the methods of cleaving DNA

Answer 16

mechanical shearing - create truly random library but ends should be modified before ligating

partial digestion with restriction enzymes

Question 17

Explain the partial digestion with restriction enzymes process

What does the degree of digestion to be done depends on?

Answer 17

enzyme that cuts frequently is used

partial digestion form overlapping fragments

on the req. size to get max no. of fragments

Question 18

Give an example for partial digestion with enzymes

Answer 18

with Sau3A generates GATC sticky ends

can be inserted into vectors cleaved at a BamHI site

Question 19

Why is it important to have overlapping fragments?

Answer 19

it increase the probability of all the sequences in genomic DNA is represented in the library

Question 20

Using which methods DNA fragments are size fractionated

Answer 20

agarose gel

density gradient cetrifugation

Question 21

Give an example for a viral vector

Answer 21

EMBL-3

Question 22

Characteristics of EMBL-3(Arms)

efficiency?

Answer 22

left arm - encode virion proteins
right arm - ori, major promoters & other essential genes

central fragment flanked by polylinker

10^8 infective phages/ ug of lambda dna
10^66 recomb./ug

Question 23

What is the best forms of dna for invitro packaging in embl-3

Answer 23

linear lambda dna

concatenated dna

Question 24

explain the process of making a human genomic library using embl-3

Answer 24

partial digestion of human dna - sau3a

lambda dna cut with bamHI

mix and seal with dna ligase

package with in-vitro phage assembly

infect into bacteria

culture in a plate

phage clone in plaques

Question 25

How to construct a cDNA library?

Answer 25

Only the eukaryotic mRNAs have an poly-A tail

so oligo dT primer is added and annealed to the tail

viral reverse transcriptase synthesize the cDNA. A terminal hairpin loop is formed

Add NaOH to remove RNA

DNA polymerase is added to extend the hairpin loop

hairpin loop is cleaved by S1 nuclease

resulting ds cDNA is cloned into a vector

Question 26

Where a cDNA library differ from genomic libraries?

Answer 26

Sequence representation

gene sequence architecture

application of screening methods

Question 27

Genomic vs cDNA in sequence representation

Answer 27

GL - Contains sequences representing the whole genome

CL - will be in some and depleted in others

Question 28

Genomic vs cDNA in Gene sequence architecture

Answer 28

GL: only the expressed dna is present - no introns, regs, intergenic

clones are smaller

Question 29

Genomic vs cDNA in screening methods

Answer 29

GL: screened by hybridization

CL: hybridization & screening methods

Question 30

How to screen a phage library with a NA probe?

probe can be?

Answer 30

aliquot of library is mixed with bacteria

plated out

NC filter is placed on agar surface

peel off the filter

it is treated with reagents to strip off phage proteins & denature DNA

pre-hybridized - denatured salmon sperm dna

hybridized with labeled denatured probe

wash filter. remove,

• unbound probe
• non specifically bound probe

expose to xray film

orient the film with the original plate and identify & pick the corresponding plaque

large scale preparation

-part of a cDNA, gene from another closely related org.