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.