Genomic DNA Library

7/28/2019 Genomic DNA Library

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What is it?

A bank or collection of genomic DNAfragments representing the entire genome of

an organism / cell


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Why genomic library?

Helps in determining the actual sequence of a geneincluding the intron/s.

Helps in positioning the gene in a chromosomal map.

Helps in identifying all the non-transcribable elements(such as promoters, UAS, enhancers).

Useful for genetic mapping.


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Important considerations

Should represent entire genome

Fragments should be of uniform size.

Large fragments are underrepresented due to

cloning bias.

Should contain (preferably) overlapping

fragments

Helps in chromosome walking


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Minimum clone number

Probability of having any given sequence in a library can be

calculated from the equation

N = ln(1-P)

ln(1-f)

P-desired probability

f- proportion of the genome in a single recombinant

f= size of insert (Kb) / size of genome (Kb) n the ratio of the size of the genome to the size of a single

insert

N-necessary number of recombinants


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Example:

To achieve probability of 99% (P-0.99)

of having a given DNA sequence represented in a library of 20Kb

fragments of mammalian genome (3x109 bp)

N = ln(1-P)

ln(1-f)

N = ln(1-0.99) = 6.9x105

ln(1- 2x104)

3x109

For library of 100 kb fragments

N = ln(1-0.99) = 1.3x105

ln(1- 1x105)

3x109

Number of clones is inverse to fragment size


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Vector capacities

Vector type Cloned DNA (kb)

Plasmid 20

phage 25

Cosmid 45

P1 phage 100

BAC 300

YAC 1000


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phage vectors

Two types

1. Insertion and2. Replacement

Both are ideal systems for genomic DNAlibrary synthesis


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BACs

Vectors that enable artificial chromosomes to be created and

cloned into E. coli.

Features:

1. Useful for cloning up to 200 kb, but can be handled like

regular bacterial plasmid vectors.

2. Useful for sequencing large stretches of chromosomal DNA;

frequently used in genome sequencing projects.

3. Like other vectors, BACs contain:

1. Origin (ori) sequence derived from an E. coliplasmid

called the F factor.

2. Multiple cloning sites (restriction sites).

3. Selectable markers (antibiotic resistance).


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Genomic DNA

Should of good quality, without

contaminations of proteins/ polysaccharides

/ phenol.

There should be no shearing. It should intact

and of high molecular weight.


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Methods for fragmentation of DNA

3 ways to make a genomic library:

1. Complete digestion (at all relevant restriction sites)

1. Produces a large number of short DNA clones.2. Genes containing two or more restriction sites may be

cloned in two or more pieces. Good for small genes or

genes with less introns.

2. Mechanical shearing

1. Produces longer DNA fragments.

2. Ends are not uniform, requires enzymatic modification

before fragments can be inserted into a cloning vector.


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3. Partial digestion

1. Cut at a less frequentrestriction site and limitthe amount and time the

enzyme is active.2. Results in population of

large overlappingfragments.

3. Fragments can be size

selected by agaroseelectrophoresis.

4. Fragments have sticky endsand can be cloned directly.

A partial digest


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Other considerations

A 4 base cutter with compatible ends for other

restriction sites is usually chosen for digestion

(For eg. Mbo I and Sau 3, both compatible to BamHI producing GATC ends)

The 5 ends of the donor DNA is dephosphorylatedwith CIAP. This prevents self ligation.


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Screening


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Screening

Documents

Genomic DNA Library