Strategies for gene isolation Flashcards
What is done in DNA cloning?
amplification of donor DNA using a suitable replicon
Give examples for types of cloning vectors?
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.
What are the properties of an ideal vector?
episomal - dont integrate into the host genome
replicates autonomously
marker
versatile - unique restriction sites, ancillary functions
high copy number
What are the 3 major steps involved in cloning?
Ligate donor dna to the vector
transfer recombinant dna to the cells
plate out cells
isolate cloned dna
What are the 2 types of DNA libraries?
genomic
cDNA - from mRNA by reverse transcriptase
How to isolate a gene?
construct a library and screen for it
What is a genomic library?
a collection of recombinant clones that together contain all the DNA present in an organism.
Properties of an ideal genome library?
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
In general what is the size of a library of a complex eukaryotic genome
10^5 - 10^6
lambda phage characteristics
size - 48.6kb
~40% not essential for propagation of phage
Types of lambda phage vectors? explain each.
Insertional ~ 12kb
substitutional ~ 23kb
Why can’t just naked DNA be inserted into bacteria? Solution?
transfection
efficiency is low(okay for small genomes)
phage DNA is packaged into capsids before introduction into bacteria
On what the no. of recombinants required to make a genomic library depends on?
genome size of the organism
size of cloned fragments
Note the equation used to calculate the no. of clones required
N = ln(1-p)/ln(1-x/y)
Properties of donor DNA to be cloned and what to look for?
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
Explain the methods of cleaving DNA
mechanical shearing - create truly random library but ends should be modified before ligating
partial digestion with restriction enzymes
Explain the partial digestion with restriction enzymes process
What does the degree of digestion to be done depends on?
enzyme that cuts frequently is used
partial digestion form overlapping fragments
on the req. size to get max no. of fragments
Give an example for partial digestion with enzymes
with Sau3A generates GATC sticky ends
can be inserted into vectors cleaved at a BamHI site
Why is it important to have overlapping fragments?
it increase the probability of all the sequences in genomic DNA is represented in the library
Using which methods DNA fragments are size fractionated
agarose gel
density gradient cetrifugation
Give an example for a viral vector
EMBL-3
Characteristics of EMBL-3(Arms)
efficiency?
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
What is the best forms of dna for invitro packaging in embl-3
linear lambda dna
concatenated dna
explain the process of making a human genomic library using embl-3
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
How to construct a cDNA library?
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
Where a cDNA library differ from genomic libraries?
Sequence representation
gene sequence architecture
application of screening methods
Genomic vs cDNA in sequence representation
GL - Contains sequences representing the whole genome
CL - will be in some and depleted in others
Genomic vs cDNA in Gene sequence architecture
GL: only the expressed dna is present - no introns, regs, intergenic
clones are smaller
Genomic vs cDNA in screening methods
GL: screened by hybridization
CL: hybridization & screening methods
How to screen a phage library with a NA probe?
probe can be?
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.
