Genomic Libraries

Learning Objectives

• Differentiate - genomic and cDNA library.

• Explain briefly the principles and steps involved in constructing genomic and cDNA libraries.

• Discuss the advantages and disadvantages on genomic and cDNA libraries.

Challenges in cloning a gene

• Target genome is more complex

• How to isolate a single gene out of thousands?

• The target gene sequence is diluted over a million fold by other genes and unwanted genomic DNA.

• To sift rapidly the large numbers of unwanted sequences to identify the particular target gene.

Libraries

• Gene library: a collection of different DNA sequence from an organism, each of which has been cloned into a vector for ease of purification, storage and analysis.

– Ideally contains at least one copy of every DNA sequence.

Genomic libraries

cDNA libraries

Gene library

(made from genomic DNA)

(made from cDNA- copy of mRNA)

What is a genomic library?

• A genomic library is a collection of DNA from a single organism,

– ideally though not necessarily containing its entire genomic DNA sequence.

• The DNA from the source organism of interest is divided into multiple fragments

• Packaged within cloning vectors such that each carries a portion of it. • Packaged within cloning vectors such that each carries a portion of it.

• The vector DNA can then be inserted into host organisms - commonly a population of bacteria - for amplification and retrieval.

Representation

• A good library should cover the whole genome which depends on,

– Size of the genome

– Size of fragment cloned

– Certainty of containing a unique sequence

– Randomness

• For example,

• if the whole genome is 2.8 x 106 kb, and if the average clone size is 20 kb, • if the whole genome is 2.8 x 10 kb, and if the average clone size is 20 kb,

– Number of recombinants should at least (n) = 1.4 x 105

Genomic Libraries

• Fragments ligated into cloning vectors

– Small insert

• Lambda phage: 20-50 Kbp

• Plasmid: ~12 Kbp

– Large insert

• BACs (Bacterial Artificial Chromosomes) 100-300 kb

• YACs (Yeast Artificial Chromosomes) ~ 500 - 1000 kb• YACs (Yeast Artificial Chromosomes) ~ 500 - 1000 kb

VectorMaximum

Insert size

Approx. No. of

clones required

in library

Advantages Disadvantages

plasmid 0 - 10 kb 107

easy to construct

libraries very many clones

requiredrelatively stable

inserts

lambda 20 kb 5 × 105

easy to construct

librariesmany clones required

relatively stable

inserts

hard to prepare DNA

from clones

easy to construct

cosmid 45 kb 2 × 105

easy to construct

librariesnot always stable

easy to prepare DNA

from clones

YAC 1 Mb 104 few clones required

very prone to

rearrangement,

difficult to construct

BAC >500 kb 5 × 104

few clones requiredsingle copy origin of

replication therefore

harder to prepare

large amounts of DNAvery stable

BACs and YACs

• BACs can hold up to 300 kb.

• The F factor of E.coli is capable of handling large segments of DNA.

• Recombinant BACs are introduced into E.coli by electroportation. Once in the cell, the rBAC replicates like an F factor.

• Has a set of regulatory genes, OriS, and repE which control F-factor replication,

• YACs can hold up to 500 kb.

• YACs are designed to replicate as plasmids in bacteria when no foreign DNA is present. Once a fragment is inserted, YACs are transferred to cells, they then replicate as eukaryotic chromosomes.

• YACs contain: a yeast centromere, two yeast telomeres, a bacterial origin of • Has a set of regulatory genes, OriS, and

repE which control F-factor replication, and parA and parB which limit the number of copies to one or two.

• A chloramphenicol resistance gene, and a cloning segment.

yeast telomeres, a bacterial origin of replication, and bacterial selectable markers.

• YAC plasmid � Yeast chromosome

• DNA is inserted to a unique restriction site, and cleaves the plasmid with another restriction endonuclease that removes a fragment of DNA and causes the YAC to become linear. Once in the cell, the rYAC replicates as a chromosome, also replicating the foreign DNA.

How to make a genomic library?

total genomic DNA

oriori

ori

ampR

ampR

restrictionenzyme

annealand ligate

genomic DNA

plasmid (black)

ampR ori ampR ori

oriori

ampR

ampR

samerestrictionenzyme

and ligate

transform E. coli;select for

Amp resistance

Genomic Library

Advantages

• Allows one to clone the entire gene, including introns and even regulatory sequences associated with the gene

• If mRNA is low abundance this may be the only method of cloning a gene

Disadvantages

• The library is very large - searching for a gene is like searching for a needle in a haystack

• Isolated genes are very large and hence more difficult to manipulate.

• Need to trim extraneous sequences

Complementary DNA library

• cDNA libraries are generated by reverse transcription of mRNA population.

• cDNA is the representative of the mRNA population.

• Cloned cDNAs lack introns and other non-coding sequences present in the corresponding DNA.

The size of the cDNA clone is significantly smaller than that of the • The size of the cDNA clone is significantly smaller than that of the corresponding genomic clone.

• cDNA library is representative of the mRNA population from which it was derived and so it varies with type of tissue, developmental stage and time.

Making a cDNA library

Step 1: Isolate RNA

• RNA is purified from tissue or cell line

• The mRNA is then isolated away from ribosomal and tRNAs

stationary supportpolyT

mRNApolyA

tissue or cell

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

• Column with oligo dT is used to bind poly A

polyTpolyA

Step 2: Obtain cDNA from RNA

• mRNA is treated with the enzyme reverse transcriptase (RT)

• The enzyme copies sequence of mRNA into first strand of DNA

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458

• Digest RNA with RnaseH

• Another enzyme (RT) is used to make second strand of cDNA

Step 3: Transformation

• Double-stranded cDNA is inserted into cloning vector

• cDNA is ligated into cloning vector (plasmid or phage)

• Vector is transformed or infected into bacteria

© 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey

07458

E. Colibacteria

plasmid

cDNA Library

• cDNA library will contain different clones where the genes is differentially spliced representing alternatively spliced variants.

• Typically, x105 clones is sufficient for the isolation of low abundance mRNAs from most cell types.

• Efficiency can be further enriched by size fractionation and testing for the desired molecule. desired molecule.

• The abundance of a particular mRNA varies with the type of tissue and proportional to the expression of that particular protein.

• There are moderate and low abundant mRNAs which necessarily requires a cDNA library.

cDNA Library – Advantages and Constraints

Advantages

• Library is small and hence much easier to screen.

• Isolated gene is small and hence easy to manipulate.

• Isolated sequence devoid of introns.

Constraints

• If gene expression is less than 1% of total RNA it is difficult to construct cDNA libraries.

• degradation of mRNA during preparation of mRNA and conversion to cDNA.

• cDNA is not a complete gene (only coding seq)

Differences between a genomic and cDNA library

Genomic Library

• Promoters

• Introns

• Intergenic

• Non-expressed genes

cDNA Library

• Expressed genes• Transcription start sites • Open reading frames (ORFs) • Splice points

Today we learnt about:

• Genomic and cDNA library.

• The principles and steps involved in constructing genomic and cDNA libraries.

• Advantages and disadvantages of genomic and cDNA libraries.

Next Day

• Screening and Enrichment of Libraries