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Genomic DNA Library

A genomic library is a collection of DNA fragments that represent an organism's entire genome. It is useful for determining gene sequences, positioning genes on chromosomes, and identifying regulatory elements. Important considerations for a genomic library include representing the entire genome with uniformly sized, preferably overlapping fragments cloned into a vector. The necessary number of clones can be calculated based on fragment size and genome size to achieve a desired probability, such as 99%, of containing a given sequence. Common vector types for genomic libraries include plasmids, phages, cosmids, BACs, and YACs that can clone inserts of different sizes.

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111 views15 pages

Genomic DNA Library

A genomic library is a collection of DNA fragments that represent an organism's entire genome. It is useful for determining gene sequences, positioning genes on chromosomes, and identifying regulatory elements. Important considerations for a genomic library include representing the entire genome with uniformly sized, preferably overlapping fragments cloned into a vector. The necessary number of clones can be calculated based on fragment size and genome size to achieve a desired probability, such as 99%, of containing a given sequence. Common vector types for genomic libraries include plasmids, phages, cosmids, BACs, and YACs that can clone inserts of different sizes.

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plastioid4079
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© Attribution Non-Commercial (BY-NC)
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What is it?

A bank or collection of genomic DNA fragments representing the entire genome of an organism / cell

Why genomic library?


Helps in determining the actual sequence of a gene including 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.

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

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

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) ln(1- 2x104) 3x109 = 6.9x105

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

Vector capacities
Vector type
Plasmid
phage Cosmid P1 phage BAC YAC

Cloned DNA (kb)


20
25 45 100 300 1000

phage vectors
Two types 1. Insertion and 2. Replacement
Both are ideal systems for genomic DNA library synthesis

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. coli plasmid called the F factor. 2. Multiple cloning sites (restriction sites). 3. Selectable markers (antibiotic resistance).

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.

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.

3. Partial digestion
1. Cut at a less frequent restriction site and limit the amount and time the enzyme is active. 2. Results in population of large overlapping fragments. 3. Fragments can be size selected by agarose electrophoresis. 4. Fragments have sticky ends and can be cloned directly.

A partial digest

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 Bam HI producing GATC ends) The 5 ends of the donor DNA is dephosphorylated with CIAP. This prevents self ligation.

Screening

Screening

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