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
(made from genomic DNA)
Gene library

cDNA libraries
(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.

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,
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

Vector

plasmid

Maximum
Insert size

0 - 10 kb

Approx. No. of
clones required
in library
107

lambda

20 kb

5 105

cosmid

45 kb

2 105

YAC

1 Mb

104

Advantages
easy to construct
libraries
relatively stable
inserts
easy to construct
libraries
relatively stable
inserts
easy to construct
libraries
easy to prepare DNA
from clones
few clones required
few clones required

BAC

>500 kb

5 104
very stable

Disadvantages

very many clones

required
many clones required
hard to prepare DNA
from clones
not always stable
very prone to
rearrangement,
difficult to construct
single copy origin of
replication therefore
harder to prepare
large amounts of DNA

BACs and YACs

BACs can hold up to 300 kb.

YACs can hold up to 500 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.

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.

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.

YACs contain: a yeast centromere, two

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

A chloramphenicol resistance gene, and a

cloning segment.

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?

ori

total genomic DNA

ampR

genomic
DNA

restriction
enzyme
anneal
and ligate
plasmid (black)
ampR

ori

ampR
same
restriction
enzyme

ori

ampR

ori

ori
ampR

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
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

Column with oligo dT is used to

bind poly A

tissue or cell

mRNA
polyA

stationary support
polyT

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

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

Digest RNA with RnaseH

Another enzyme (RT) is used to

make second strand of cDNA

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

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

plasmid

E. Coli
bacteria

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

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.

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