Construction Of Genomic Library

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

compounds,

or

contaminated restriction

with

enzyme

polysaccharides,

resistant

DNA

phenolic

samples

successfully used to generate libraries. BAC vector •

Insert size-125 kb to 200kb

•

100,000 to >200,000 clones (based on required x coverage)

are

•

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 colorectal 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 expressionvector 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 noncoding 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.