GENOME LIBRARY CONSTRUCTION

cDNA LIBRARY

WHAT IS GENOME?
Life is specified by genomes. Every organism, including humans, has a
genome that contains all of the biological information needed to build
and maintain a living example of that organism. The biological
information contained in a genome is encoded in its deoxyribonucleic
acid (DNA) and is divided into discrete units called genes. Genes code for
proteins that attach to the genome at the appropriate positions and
switch on a series of reactions called gene expression

Genomic Library Construction

Custom Genomic Library Construction Service is offered in BAC, cosmid,

bacteriophage or plasmid vectors. High molecular weight Pulse Field Gel
Electrophoresis (PFGE) isolated DNA is digested, CIP treated and size
fractionated. The size fractionated DNA is ligated to a suitable vector
package and is harvested.

Bionexus can construct genomic libraries from nanogram quantities of

genomic DNA or milligram quantities of the tissue. Bionexus routinely
generates libraries from trace amount of genomic DNA, chromosomes,
uncultured environmental microbes, or base-modified phages. Highly
methylated DNA contaminated with polysaccharides, phenolic
compounds, or restriction enzyme resistant DNA samples are
successfully used to generate libraries.

BAC vector

• Insert size-125 kb to 200kb

• 100,000 to >200,000 clones (based on required x coverage)
 • Very cost effective
• Turnaround time 6-8 weeks

Cosmid vector

• Insert size 30 kb to 40 kb
• >107 primary clones
• 90-95% recombinants
• Turnaround time 4 weeks

Bacteriophage / Plasmid vector

• Insert size 9-23kb (bacteriophage vector) and 2-10 kb (plasmid

vector)
• >107 primary clones
• 90-95% recombinants
• Supplied as amplified or un-amplified library
• Turnaround time 4 weeks

Clones from all the libraries can be arrayed on nylon membranes.The

libraries will be amplified once to render stability to the clones and will
be titrated and supplied in SM buffer. A complete report containing the
specifications of the library and other data will be provided along with
the libraries.

*Gridding available only with libraries in plasmid vectors.

Estimated Delivery Date: The Genomic Library would require 4 weeks to

complete after receiving
starting materials (custom vector will require additional time). Client will
be the sole owner of the
Genomic Libraries, RNA, DNA, and all sequence data generated.
BIONEXUS is providing a service. The estimated time required to
complete your project is noted above. The project starting date will be
finally determined as soon as we receive the signed quotation and all
starting materials at our facility

INTRODUCTION:
The recombinant DNA field is every green field in biology. Now it has
more advanced techniques. One of the main techniques is cDNA library
construction and it’s the basic step in rDNA. So now we see about what
is cDNA, Construction methods and uses. Central dogma states that
biological information goes from DNA to RNA to protein

Figure 1. Central dogma: DNA to RNA to mRNA to protein. Coding

sequence (purple) exons are spliced together and the 5' cap and 3' polyA
tail is added to produce a mature mRNA molecule from the primary
transcript. The mRNA is translated into protein.

However, there are times when information goes from RNA to DNA.
Viruses such as HIV have RNA genomes that can be converted into DNA
by an enzyme called reverse transcriptase. Molecular biologists realized
that they could use reverse transcriptase to convert mRNA into
complementary DNA and thus was born the term cDNA. The one
difference between eukaryotic and prokaryotic genes is that eukaryotic
genes can contain introns (intervening sequences), which are not coding
sequences, and must be spliced out of the RNA primary transcript before
it becomes mRNA and can be translated into protein. Prokaryotic genes
have no introns, so their RNA is not subject to splicing.

Often it is desirable to express eukaryotic genes in prokaryotic cells. A

simplified method of doing so would include the addition of eukaryotic
DNA to a prokaryotic host, which would transcribe the DNA to mRNA
and then translate it to protein. However, as eukaryotic DNA has introns,
and since prokaryotes lack the machinery to splice them, the splicing of
eukaryotic DNA must be done prior to adding the eukaryotic DNA into
the host. This DNA which was made as a complementary to the RNA is
called complementary DNA (cDNA). To obtain expression of the protein
encoded by the eukaryotic cDNA, prokaryotic regulatory sequences
would also be required (e.g. a promoter).

What is cDNA?
Complementary DNA (cDNA) is DNA synthesized from a mature
mRNA template in a reaction catalyzed by the enzyme reverse
transcriptase. The cDNA is made from mRNA with the use of a special
enzyme called reverse transcriptase, originally isolated from retroviruses.
Using an mRNA molecule as a template, reverse transcriptase
synthesizes a single-stranded DNA molecule that can then be used as a
template for double-stranded DNA synthesis. cDNA does not need to be
cut in order to be cloned.

Why we construct cDNA.

 cDNA is a more convenient way to work with the coding
sequence than mRNA because RNA is very easily degraded by
omnipresent RNases. This the main reason cDNA is sequenced rather
than mRNA. Likewise, investigators conducting DNA microarrays often
convert the mRNA into cDNA in order to produce their probes. Let's see
what is required to produce cDNA.

Basic reagents for cDNA library construction:

By definition, cDNA is double-stranded DNA that was derived from

mRNA which can be obtained from prokaryotes or eukaryotes. Once the
mRNA is isolated, you need a few more reagents: dNTPs (dGTP, dCTP,
dATP and dTTP), primers, and reverse transcriptase which is a DNA
polymerase (figure 2). Mix the mRNA with the other reagents and allow
the polymerase to make a complementary strand of DNA (first strand
synthesis). Next, the mRNA must be removed and the second strand of
DNA synthesized. There are many technical details in these steps, but we
do not need to focus on them at this time.

Figure 2. Four basic reagents needed to produce cDNA: mRNA as

template, dNTPs, reverse transcriptase and primers.

The only issue worth mentioning now is that three different types of
primers can be used (figure 3). 1) If the mRNA has a poly-A 3' tail, then
an oligo-dT primer can be used to prime all mRNAs simultaneously. 2) If
you only wanted to produce cDNA from a subset of all mRNA, then a
sequence-specific primer could be used that wil only bind to one mRNA
sequence. 3) If you wanted to produce pieces of cDNA that were scattered
all over the mRNA, then you could use a random primer cocktail that
would produce cDNA from all mRNAs but the cDNAs would not be full
length. The major benefits to random priming are the production of
shorter cDNA fragments and increasing the probability that 5' ends of
the mRNA would be converted to cDNA. Because reverse transcriptase
does not usually reach the 5' end of long mRNAs, random primers can be
beneficial.

Figure 3. Three ways to prime the production of cDNA: oligo-dT primer

(red), sequence-specific primer (green), random primer (blue).

Random Priming Technique

One of most frequently cited papers is one by Feinberg and Vogelstein

(1983). Although Voglstein has dissected the molecular pathway to colo-
rectal cancer and discovered many other fundamental biological
processes, this technique paper ishas been cited by almost every
molecular biologist at one point or another. The reason for its popularity
is the simple solution to a vexing problem. How can you produce a
complementary strand of DNA when you don't know the sequence or you
want to produce many short DNA copies of every section of DNA in a
complex mixture?

The solution is the random primer which is so simple that it left many
people asking, "Now why didn't I think of that?". Random primers are
short segments of single-stranded DNA (ssDNA) called oligonucleotides,
or oligos for short. These oligos are only 8 nucleotides long (octamers)
and they consist of every possible combination of bases which means
there must be 48 = 65,536 different combinations in the mixture.
Because every possible hexamer is present, these primers can bind to
any section of DNA.

Figure 1. Three examples of hexamers from the mixture of all possible

hexamers in random primers. These three particular primers could bind
to three overlapping portions of this mRNA to prime the production of
cDNA. The primer that arrives first will bind and the other two will have
to find another segment of DNA (either another copy of the same mRNA
or from a different locus) to bind. Hexamers were used instead of
octamers to minimize clutter in the figure.

The only other point to consider is that their short length means that
they do no bind to a segment of ssDNA with much force since there are
very few hydrogen bonds holding the two strands together (template and
oligo). Nevertheless, the method works amazingly well and is still in use
to produce random pieces of DNA for probe production. These probes can
be used on blots or DNA microarrays.

SYNTHESIS OF COMPLIMENTRY DNA:

Construction of cDNA library:

USES OF cDNA LIBRARY:

The immune response of patients with paraneoplastic neurological

degeneration (PND) involves the generation of high-titre antibodies
against neuronal antigens. These antibodies were originally used to
characterize the target antigens through immunohistochemistry and
western blotting. They have also been identified through expression-
vector complementary DNA cloning (diagram). In this technique, a cDNA
library is expressed by a bacteriophage, with each colony expressing a
single cDNA. A single plate of bacteriophage can harbour up to 105
different cDNA clones. The expressed cDNAs are transferred to
nitrocellulose and can then be probed with patient antisera. Many PND
antigens were identified in this manner.
ANALYSES OF cDNA LIBRARY
The genetic material of the cell is composed of Nucleic Acids. These can
be separated into two forms: deoxyribo-nucleic acids (DNA) which make
up the chromosomes; and ribo-n ucleic acids (RNA) which decode the
genes encoded in the chromosomal DNA and use the information to
produce proteins for the cell. When a gene is activated (i.e. made
available for usage), an enzyme called RNA polymerase makes an RNA
copy of the gene (called an hnRNA; hn is for heavy, nuclear), which is
then processed into a more compact form (called mRNA; m is for
messenger) that exits the nucleus and is used as a template for protein
production. One of the major differences between hnRNA and mRNA is
the existence of introns. Introns are present in chromosomes as non-
coding stretches of DNA which break up individual genes into small,
separated fragments, called exons. When RNA polymerase transcribes a
gene, it copies the introns and exons together, so that the resulting
hnRNA contains the fragmented gene plus all of its introns. A group of
RNA-protein enzymes (called snRNP's) attach to the introns in hnRNA's
to form Spliceosomes, which excise the introns and splice the exons
together to form the entire, uninterrupted gene. After other
modifications, the result is an intronless mRNA copy of the gene.

The only problem with mRNA is that, for various reasons, it is much
more difficult to work with, in the laboratory, than DNA. Fortunately, all
RNA viruses (including Poliovirus, Herpesvirus, HIV, and many more)
produce an enzyme called Reverse Transcriptase (RT) which makes DNA
copies of RNA strands and is easy to mass produce from bacterial
cultures. Because the DNA is a copy of an RNA, rather than vice versa, it
is called cDNA (c is for copy). The most common usage of RT is to make
cDNA from mRNA. cDNA has two advantages over chromosomal DNA:
there are no introns, so it is easier to identify and characterize the genes;
and cDNA only represents those genes that are being actively used by the
cell, since RNA polymerase only transcribes activated genes.

Now for the "library". If you have a piece of a gene and you want the rest
of the gene, it would take a very long time to search from one end of a
genome to the other looking for your gene. On the other hand, if you
divide the genome into fragments, and then identify which fragment
contains your gene, it takes very little time to search from one end of a
fragment to the other. This is essentially what libraries are about. To
make a library, you divide a large pool of DNA into smaller units, and
then give each unit the ability to replicate independently, by splicing it
into a vector (like a virus or an artificial chromosome), and cloning it into
a cell which will reproduce and make copies. Genomic libraries exist for
all organisms commonly used in the lab, and consist of enzymatically
digested chromosome fragments spliced into various vectors and placed
in various cells depending on the size of the fragments (phage libraries in
bacteria for small fragments to YAC libraries in yeast for huge
fragments).

cDNA libraries are simpler to construct, because cDNA's, like their

parental mRNA's, are already fairly short, so an entire cDNA can be
spliced into a single vector. The reason you need to make a library is that
cells produce tens of thousands of different mRNA's at a time, so that
after using RT to make cDNA, you still have a massive pool of different
cDNA's with which to work. As stated above, cDNA libraries have
advantages over genomic libraries: there are no introns, so there is no
danger of pieces of your gene being chopped onto separate clones; and
the library is (hopefully) enriched for your gene, since instead of one or
two copies, as in the genomic library, you have as many copies as the cell
could produce mRNA's for that gene. So most molecular biologists, when
searching for a new gene, start by screening a cDNA library from a tissue
or organism that they suspect is actively using that gene. Most new
genes are found this way.