2. DEFINITION WHAT IS GENOME?
Genome is nothing but it 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.

3. WHAT IS GENOMIC LIBRARY?
A genomic library is a population of host bacteria, each of which carries a DNA molecule that was inserted into a cloning vector, such that the collection of cloned DNA molecules represents the entire genome of the source organism.

4. Cloning Vectors
A vector is used to amplify a single molecule of DNA into many copes. A DNA fragment must be inserted into a cloning vector. A cloning vector is a DNA molecule that has an origin of replication and is capable of replicating in a bacterial cell.
Most vectors are genetically engineered plasmids or phages. There are also cosmid vectors, bacterial artificial chromosomes, and yeast artificial chromosomes.

5. Plasmid Cloning Vectors
Plasmids are circular, double-stranded DNA molecules that exist in bacteria and in the nuclei of some eukaryotic cells.
They can replicate independently of the host cell. The size of plasmids ranges from a few kb to near 100 kb
Can hold up to 10 kb fragments
Plasmids have an origin of replication, antibiotic resistance genes as markers, and several unique restriction sites.

6. Phage Cloning Vectors Cosmid Cloning Vectors
Lambda is most common phage
Fragments up to 23 kb can be may be accommodated by a phage vector
Cosmid Cloning Vectors
Fragments from 30 to 46 kb can be accommodated by a cosmid vector.
Cosmids combine essential elements of a plasmid and Lambda systems.

7. Bacterial Artificial Chromosomes(BACs)
BACs can hold up to 300 kbs.
The F factor of E.coli is capable of handling large segments of DNA.
A chloramphenicol resistance gene, and a cloning segment.
Yeast Artificial Chromosomes(YACs)
YACs can hold up to 500 kbs.
YACs contain: a yeast centromere, two yeast telomeres, a bacterial origin of replication, and bacterial selectable markers

8. Genomic library Made from fragments of genomic DNA
Genomic DNA cut up with restriction enzymes or randomly broken by mechanical shearing
Fragments ligated into cloning vectors
Small insert
Lambda phage
Plasmid
Large insert
BACs
Genomic libraries, as their name indicates, are made up of fragments of genomic DNA. To obtain fragments of the right size for cloning, the genomic DNA is either cut with restriction enzymes or randomly broken. Random shearing can be achieved by forcing chromosomal DNA through a small orifice, like a syringe needle, or by sonication. Genomic fragments obtained either by restriction-enzyme digestion or by mechanical shearing are then ligated into a cloning vector. There are a variety of cloning vectors that differ in the size of the DNA fragment that they can accommodate. Smaller inserts in the range of 20–40 kb can be cloned into plasmid or lambda phage vectors, while large inserts on the order of 100–300 kb can be cloned into vectors such as bacterial artificial chromosomes (BACs). Even larger inserts up to 1 MB can be accommodated in yeast artificial chromosomes (YACs).

9. How to make a genomic library
ori
total genomic DNA
ampR
genomic DNA
restriction
enzyme
anneal
and ligate
ori
ampR
ori
plasmid (black)
This schematic depicts the construction of a genomic library in a plasmid vector. The top left shows genomic DNA being cut with restriction enzymes to produce different fragments (shown as different colored bars). The bottom left part of the schematic shows the plasmid cloning vector being cut with the same restriction enzyme as the genomic DNA was (to yield compatible, or “sticky,” ends). The genomic fragments are then mixed with the plasmid vector in the presence of ligase. This operation results in ligation of a different genomic fragment into each copy of the cloning vector. The resulting circular DNA is then amplified by transforming it into bacteria. Plating of the bacteria on medium containing the antibiotic allows only those bacterial cells which have taken up the plasmid carrying an antibiotic resistance gene to grow. In the case of mechanically sheared DNA, there is an additional step in which synthetic linkers are added to make the ends compatible with the cloning vector.
ori
ori
ampR
ampR
ampR
same
restriction
enzyme
transform E. coli;
select for
Amp resistance

10. WHAT IS cDNA?
cDNA is DNA synthesized from a mature mRNA template in a reaction catalyzed by the enzyme reverse transcriptase.

11. cDNA strand synthesis

12. WHAT IS cDNA LIBRARY?
A cDNA library is a collection of cloned cDNA (complementary DNA) fragments inserted into a collection of host cells, which together constitute some portion of the transcriptome of the organism. cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an organism.

13. WHY WE CONSTRUCT cDNA LIBRARY?
A genomic library represents all the DNA in the genome, whether it is expressed or not. However, very often it is really the genes that are being expressed that are our main target.
Finally, if we base our library on the mRNA extracted from the target cells, rather than on their DNA, we will be able to focus more closely on the real target, and make the identification of the required clones much more efficient.
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.

14. WHAT ARE THE BASIC REAGENTS NEED FOR cDNA LIBRARY CONSTRUCTION?
Four basic reagents needed to produce cDNA: mRNA as template, dNTPs, reverse transcriptase and primers.

15. Different types of primers:
If the mRNA has a poly-A 3’tail,then an oligo-dt primer can be used to prime all mRNA simultaneously.
If you only wanted to produce cDNA from a subset of all mRNA, then a sequence-specific primer could be used that will only bind to one mRNA sequence.
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.

16. Making a cDNA library Step 1: Isolate RNA
RNA is purified from tissue or cell line
The mRNA is then isolated away from ribosomal and tRNAs
Column with oligo dT is used to bind poly A
tissue or cell
mRNA
polyA
stationary support
polyT
The basis for a cDNA library is RNA that is purified from a tissue (such as heart or skin) or from a cell line. To obtain only the messenger RNA (mRNA) and get rid of non-coding RNAs such as ribosomal RNAs and tRNAs, oligo dT chromatography is used. This uses short stretches of the nucleotide thymidine that are bound to a column or to magnetic beads. The oligo dT binds to the polyA tail found on all messenger RNAs. Elution from the column results in a relatively pure population of mRNA molecules.

17. Step 2: Obtain cDNA from RNA
mRNA is treated with the enzyme reverse transcriptase
The enzyme copies sequence of mRNA into first strand of DNA
Another enzyme is used to make second strand of cDNA
The isolated mRNA is then incubated with the enzyme reverse transcriptase. This enzyme is able to copy the sequence of RNA into a complementary DNA (cDNA) strand. It requires an RNA template, a primer (usually oligo dT), and sufficient nucleotides. After a first strand of cDNA has been made, the RNA is digested away using an enzyme called Rnase H. Then another enzyme (or reverse transcriptase) is used to copy the first strand to make a second strand of cDNA.

18. Step 3: Transformation
Double-stranded cDNA is inserted into cloning vector
cDNA is ligated into cloning vector (plasmid or phage)
Vector is transformed or infected into bacteria
The double-stranded cDNA is ligated into a cloning vector. This vector is generally a small-insert plasmid or phage vector, as cDNA is rarely longer than 10 kb. The clones are then inserted into bacteria by transformation in the case of plasmids or by infection in the case of phage. In an ideal library, each colony or plaque contains a different clone that originated from a single mRNA molecule. In fact, cDNA libraries can differ greatly in quality. Not infrequently, a significant percentage of the clones in the library do not contain an insert. In other cases, the inserts can be very short, representing only partial copying of the mRNA to cDNA.
plasmid
E. Coli
bacteria

19. Screening methods for cDNA libraries
The following methods for used to screen the cDNA libraries, they are
1. Colony Hybridization
2. Screening expression libraries with Antibody or other probes

20. Colony Hybridization Colony DNA is attached to membrane
selected
colonies
Colony DNA is attached to membrane
DNA is screened with labeled probes
DNA is labeled with radioactivity
Labeled DNA is allowed to hybridize with DNA on membrane
After washing, positive hybridization spots are identified
membrane
Radioactive
probe
Libraries have several potential uses. The most common is to attempt to isolate a clone with a sequence that is either identical or similar to a known piece of DNA. Hybridization, which makes use of the complementary nature of the two strands of DNA, is used to search a library for identical or similar sequences. This process is known as screening a library. To screen a library, the DNA in each of the bacterial colonies is first attached to a nylon or nitrocellulose membrane. This result is achieved by placing the membrane over the colonies and letting them stick to it. The membrane is then removed and treated to break open the cells and attach irreversibly the DNA that is released from the cells. Probes are then labeled and hybridized to the DNA attached to the membrane (described later in this chapter). After washing and exposure to X-ray film, the location of colonies containing a specific cDNA can be visualized.
hybridization
X-ray film

21. Screening expression libraries with Antibody or other probes

22. The uses of cDNA library
There are no introns
Useful in immunological techniques(PND disorders)
Identification of tissue expression between two tissues or cell type…more..

23. cDNA Library vs Genomic Library
Total Gene
Chromosomal DNA
mRNA
Reverse transcription
Restriction digestion
Complete
gene
cDNA
Smaller
Library
Larger
Library