Transcript Genomic Library cDNA Library

BSL2016 / 2018 LEC 8 Genomic Libraries (1)
How does a genomic library differ from a cDNA library?
What is a genomic library and why is it important?
cDNA Library
Genomic Library
Only contains expressed
sequences mRNA/cDNA
Contains the entire genome
Does NOT contain tRNA
or rRNA sequences
tRNA and rRNA genes are
Present.
Does NOT contain noncoding sequences
Contains EVERYTHING
Promoters, introns, junk,
Cannot be used for mapping
Essential in genome
Sequencing projects
Cloned sequences are short
typically 1-2 kb
Cloned sequences are long
up to 100kb
BSL2016 / 2018 LEC 8 Genomic Libraries (1)
What is a genomic library and why is it important?
A genomic library is a collection of cloned sequences which
represents the entire genome.
It allows the analysis of gene promoters which control how genes
function (where and when they are expressed, and in response to
which stimuli)
It allows the creation of maps of the genome which detail which
genes are located where, and the relative distance of one gene from
the other.
Used extensively in the Human Genome Sequencing Project.
BSL2016 / 2018 LEC 8 Genomic Libraries (1)
How many clones are needed to ensure that the complete genome
has been cloned?
Depends on two factors:
1) Size of the genome; 2) Size of the cloned fragment
Species
Genome size
( bp )
No. of clones
E. coli
4 x 10 6
700
D. melanogaster
8 x 10 7
1 x 10 4
H. sapiens
3 x 10 9
5 x 10 5
P. sativum
4.5 x 10 9
1 x 10 6
Assuming that
17kb is the size
of the cloned
fragment……..
The larger the size
of the cloned
fragment, the
smaller the number
of clones.
BSL2016 / 2018 LEC 8 Genomic Libraries (1)
What factors need to be taken into account when preparing genomic
DNA for library construction?
You need to cut DNA with restriction enzymes so that a series of
overlapping genomic fragments is created :
This is essential for gene isolation and for genome mapping
BSL2016 / 2018 LEC 8 Genomic Libraries (1)
The restriction enzyme used to cleave the genomic DNA must NOT
cut in the middle of genes, otherwise those genes will be “lost”
from the library.
BSL2016 / 2018 LEC 8 Genomic Libraries (1)
Which enzyme should you use to cleave genomic DNA?
Remember that you want to clone long, overlapping fragments.
All genes will be cleaved
Complete cleavage > fragments too short
Partial cleavage > fragments too long
Fragments are too large to insert
into most cloning vectors.
BSL2016 / 2018 LEC 8 Genomic Libraries (1)
The best strategy to produce fragments which are long, and
overlapping is to choose an enzyme (Sau3A) which cuts very
frequently in the genome – not obvious!!
But only to cleave a few of these sites at random (partial cleavage)
Which will result in a population of long, randomly cleaved fragments.
BSL2016 / 2018 LEC 8 Genomic Libraries (1)
BSL2016 / 2018 LEC 8 Genomic Libraries (1)
Bacteriophage lambda is the vector of choice for the creation of
genomic libraries (apart from sequencing and mapping projects)
Head (DNA)
Tail
BSL2016 / 2018 LEC 8 Genomic Libraries (1)
Phage l genome is approx 50 kb long.
In the process of head filling, a cos
site is recognised, DNA is reeled in
until the second cos site is reached,
and is then cleaved.
Central region of the l genome is not
essential for phage functions
BSL2016 / 2018 LEC 8 Genomic Libraries (1)
These are called “replacement
vectors” because the central
non-essential (stuffer)region
of the phage genome is
replaced with genomicDNA
BSL2016 / 2018 LEC 8 Genomic Libraries (1)
Lysis of the bacterial lawn
results in “plaques”