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Recombinant DNA Libraries
Recombinant DNA Libraries
• A DNA library is a collection of DNA clones,
gathered together as a source of DNA for
sequencing, gene discovery, or gene function
studies.
– Libraries are collections of cloned DNA fragments from a
particular organism contained within bacteria or viruses as the
host
– Libraries can be saved for relatively long periods of time and
"screened" to pick out different genes of interest
• Depending on the source of the DNA there are
three types of rDNA libraries .
Recombinant DNA Libraries
1.Genomic library, a collection of clones
containing at least one copy of every DNA
sequence in a genome. Genomic libraries are
available for many organisms.
2.Chromosome libraries are collections of
cloned fragments of individual chromosomes.
3.Complementary DNA (cDNA) libraries are
cloned collections of DNA copied from a cell’s
mRNA.
Genomic Library
• Genomic library, produced when the complete genome of a
particular organism is cleaved into thousands of fragment and
all the fragments are cloned by insertion into a cloning vector.
• The first step in preparing a genomic library is isolation and
digestion of the DNA by restriction endonucleases
– This process produces fragments of DNA that include the
organism's entire genome
• A plasmid, BAC, YAC or bacteriophage vector is digested
with the same enzyme
• DNA ligase is used to ligate genomic DNA pieces and
vector
Genomic Libraries
• In theory, all DNA fragments in the genome
will be cloned into a vector
• Recombinant vectors are then used to
transform bacteria or yeast cells .
• Each transformed bacterium or yeast cell
grows into a colony, or “clone,” of identical
cells, each cell bearing the same recombinant
plasmid.
• Consider each clone a "book“ in this "library"
of DNA fragments
Genomic Libraries
 There are three general ways to produce genomic libraries:
a. Complete digestion with restriction enzyme, cleave at all
relevant restriction sites. This has drawback:
1) Genes containning one or more sites for the
restriction enzyme will be cloned into two or more
pieces.
2) To screen the entire genome, a very large number of
clones would have to be examined, because insert
DNA size is relative small.
b. Longer DNA fragments can be generated with
mechanical sheering (e.g passage through a syringe
needle) rather than restriction enzyme cutting. A
disadvantage is the absence of uniform ends, require
enzymatic modification before insertion into a clonning
vector.
Genomic Libraries
c. Partial digestion with a restriction enzyme is controlled
so that it cuts only some of the available sites. Ideally,
this results in cloninng a population of overlapping
fragments representing the entire genome .
1) Partially digested DNA molecules in a certain size
range are selected by density gradient centrifugation
or agarose gel electrophoresis.
2) DNA fragments with sticky ends from restriction
digestion can be cloned directly
3) Genomic sequences are not equally represented in
the libraries, because:
a) Regions of DNA with relevant restriction sites
very close together or very far apart are removed
at the size selection.
b) Some regions of eukaryotic DNA prevent vector
replication in E. coli, and so are eliminated from
the library.
Genomic Libraries
• One disadvantage of creating this type of library for
eukaryotic genes
– Is that nonprotein coding pieces of DNA called introns are cloned in
addition to protein coding sequences (exons)
• Because a majority of DNA in any eukaryotic organism
consists of introns
– Many of the clones in a genomic library will contain non-protein
coding pieces of DNA
• Another limitation of genomic libraries is that many
organisms, including humans, have such a large genome
– That searching for a gene of interest really is like searching for a
needle in a haystack!
cDNA Library
 mRNA from the tissue of interest is isolated and used for making the
library
 However, mRNA cannot be cut directly with restriction enzymes
• So it must be converted to a double-stranded DNA molecule
 A viral enzyme called reverse transcriptase (RT) is used to catalyze the
synthesis of single-stranded DNA from the mRNA
This enzyme is made by a class of viruses called retroviruses
– So named because they are exceptions to the usual flow of genetic
information
– Instead of having a DNA genome that is used to make RNA,
retroviruses have an RNA genome
– After infecting host cells ,they use RT to convert RNA into DNA so that
they can replicate
 Since this DNA has been synthesized as an exact copy of mRNA , it is
called complementary DNA (cDNA)
 cDNA is a DNA molecule synthesied from mRNA by reverse
transcriptase enzyme .
cDNA Libraries
 The cDNA derives only from mature mRNA. Introns are not
present.
 The poly(A) tail at the 3’ end of the mRNA is useful for:
a. Isolating mRNA from cell lysates by passage over an
oligo(dT) column.
i. The mRNA’s poly(A) tail sticks to the poly(T) attached to
the column substrate.
ii. Other molecules pass through the column, but mRNAs
are retained.
iii. mRNAs are eluted with decreasing ionic strength
buffer, resulting in significant purification.
b. Priming the synthesis of cDNA, by providing a known 5’
sequence.
cDNA Libraries
 Synthesis of cDNA involves these steps (Figure):
a. A short oligo(dT) primer is used. It anneals to the mRNA’s poly(A)
tail, allowing reverse transcriptase to synthesize cDNA. This
creates a DNA-mRNA hybrid.
b. RNase H degrades the mRNA strand, creating small RNA
fragments that serve as primers.
c. DNA polymerase I makes new DNA fragments, and DNA ligase
connects the new DNA fragments to make a complete chain.
d. The resulting cDNA is a double-stranded copy of the starting
mRNA.
The synthesis of complementary DNA (cDNA) from a polyadenylated mRNA
 A method for cloning cDNA involves :
a. Introducing restriction site linkers to the ends of the cDNA by
blunt end ligation.
b. Digestion with the cognate restriction enzyme to create sticky
ends.
c. Mixing cDNA with vector DNA cut with the same restriction
enzyme in the presence of DNA ligase.
d. Transforming into an E. coli host for cloning.
 If the cDNA has sites for the same restriction enzyme
used in the polylinker, the cDNA will be cloned in pieces.
 The problem is avoided by using polylinkers engineered
with appropriate ssDNA overhangs (sticky ends) so that
restriction digestion is unnecessary.
The cloning of cDNA using BamHI linkers
cDNA Libraries
• A primary advantage of cDNA libraries over genomic
libraries
– Is that a cDNA library is a collection of actively expressed
genes in the cells or tissue from which the mRNA was
isolated
– Introns are not cloned in a cDNA library, which greatly
reduces the total amount of DNA that is cloned compared
to a genomic library
For this reason, cDNA libraries are typically preferred over
genomic libraries when attempting to clone a gene of
interest
cDNA Libraries
• One disadvantage is that cDNA libraries can be
difficult to create and screen if a source tissue
with an abundant amount of mRNA for the
gene is not available
• But polymerase chain reaction (PCR) can
frequently solve this problem
Chromosome libraries
1. Screening the genomic library of an organism
with a large genome is laborious. Screening
time can be reduced if a gene has been
localized to a chromosome, by examining a
library made from only that chromosome.
Human, for example, have 24 different
chromosome libraries (22 autosomes, X and
Y).
2. Separating chromosome so they may be
individually cloned is accomplished with
techniques such as flow cytometry.
Library Screening
• Once either a genomic library or a cDNA library is
created
– It must be screened to identify the genes of interest
• One of the most common library screening
techniques is colony hybridization
- In colony hybridization, bacterial colonies from the library
containing recombinant DNA are grown on an agar plate
• Hybridization is a general technique for detecting a
specific gene or mRNA, by allowing a labeled probe
with the complementary sequence to bind to the
target molecule.
Probes
• Probes are either cloned or synthetic fragment of single
strande nucleic acid ( usually DNA but may be RNA ) used
in either DNA:DNA or DNA:RNA hyperdization )
• Probes are usually 20 to 30 bases long and can be labeled
with radioactive tags or fluorescent tags.
• Probe is capable of hyperdizing with a DNA fragments that
carry the complementary sequence .
• Types of probes :
- Cloned DNA probes .
- cDNA probes .
- Synthetic oligonucleotide probes .
- What is the difference between cloned probe and cDNA
probe ?
Heterologous Probes
 It is possible to identify specific genes in a
genomic library using cloned equivalent genes
(heterologous probes) from other organisms,
especially if the gene is highly conserved or
the species are closely related.(many gene
sequences in rats and mice are similar to
those found in human genes )
Synthetic Oligonucleotide Probes
1. Synthetic oligonucleotides are useful in probing libraries, sequence data
are available for part of the gene of interest. Knowledge of substitutions
produced by mutation also aids probe selection. Sequences for many
genes are available in GenBank.
2. Using the universal genetic code, the amino acid sequence is used to
design a DNA oligonucleotide probe. Degeneracy in the genetic code
means that a mixture of oligonucleotides must be prepared, each of
which encodes the target protein.
3. The library is probed with the oligonucleotide mixture to detect the gene
of interest.
Library Screening
Clones detected by the
probe are characterized
Screening a cDNA library
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One way to select a cDNA clone from the library is to detect a
protein product it is producing (Figure).
For protein to be produced, an expression vector is needed,
in which the cloned cDNA is inserted between a promoter and
a transcription terminator.
Labeled antibodies are used to detect the specific protein in a
host colony. An array of colonies is transferred to a membrane
fiter, cells are lysed and their proteins bind to the filter, which
is incubated with the relevant antibody.
Radioactively labeled antibody bound to colonies is detected
by an autoradiogram, in which the dry fiter is placed on X ray
film in the dark for a number of hours. Colonies with antibody
bound will be visible as dark spots on the film.
Once identified, the cDNA can be used .
Screening for specific
cDNA plasmids in a cDNA
Library by using an antibody probe
The antibody binds to a specific
Site on a protein that is made via
The inserted foreign DNA. This
Is a Western Blot since it uses antibody
To detect a protein.