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.