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Dna libraries

DNA libraries allow for the storage and organization of genetic information, similar to how physical libraries store books. There are two main types of DNA libraries: genomic libraries, which are created from genomic DNA and contain entire genes with exons and introns, and cDNA libraries, which are created from mRNA and contain only exons. To create a genomic library, genomic DNA is isolated, fragmented, and inserted into cloning vectors within host bacteria. For cDNA libraries, mRNA is isolated, reverse transcribed into cDNA, which is then amplified and inserted into vectors. Both library types are screened to find clones containing desired DNA sequences.

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DNA Libraries Dr Ravi Kant Agrawal, MVSc, PhD Senior Scientist (Veterinary Microbiology) Food Microbiology Laboratory Division of Livestock Products Technology ICAR-Indian Veterinary Research Institute Izatnagar 243122 (UP) India
Similar to Book collections can be stored in a library; collections of genes can be made and stored in gene libraries ! 1.Genomic libraries are made from DNA and contain entire genes (exons and introns). 2.cDNA libraries are made from mRNAs that are converted into DNA (only exons) cDNA libraries are made from mRNAs It contains only exons. Libraries
Genomic libraries 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. Genomic libraries are made from DNA and contain entire genes (exons and introns).
Construction of Genomic library 1) Isolation of chromosomal DNA 2) Fragmentation of DNA:- It is cleaved by 2 ways a) Mechanical shearing - it involves pipetting, physical agitation (vortexing), sonication. b) Restriction enzyme digestion Advantages: It produce cohesive DNA ends for efficient ligation. Choose enzymes for different average insert sizes. (e.g. 4-base cutter, 6-base cutter, longer recognition site) Disadvantages: We may get fragment of different sizes, due to random distribution of enzyme sites. Much eukaryotic DNA is extensively methylated. Enzymes may not recognize methylated DNA. We can vary time of digestion (or of physical treatment) to produce DNA fragments of desired size.
(a) Complete digestion ensures that all restriction enzyme recognition sites (RE) are cut. (b) Partial digestion results in the cleavage of a random subset of the recognition sites. Partial digestion will generate a variety of products as indicated Some RE are blunt end cutters e.g. HaeIII and AluI. The blunt ended DNA fragments are problematic while its cloning because the ligation of sticky-ended DNA is more efficient than blunt ended DNA fragment.
 Sticky ends DNA fragments are produced in 2 ways:- 1. Digestion with Restriction enzymes that generate sticky ends. 2. Addition of Linkers or adaptors.
Linkers or adaptors: The blunt ended DNA fragments can be ligated to oligonucleotides that either contain the recognition sequence for a restriction enzyme (linkers) or possess one blunt end for ligation to the genomic DNA and an overhanging sticky end for cloning into particular restriction sites (adaptors).
3) Isolation and ligation of DNA fragments:- Purify fragments of required size by agarose gel electrophoresis, and elution of fragments from gel. Ligate fragments into a vector which is pre-cut with a restriction enzyme, using DNA ligase.
cDNA libraries All cells within an individual organism are derived from the same genome sequence, but the genes expressed in cell is unique to individual cell type e.g. some of the genes expressed within a skin cell will be different to those of a muscle cell. DNA – all genes are present in every cell Only some genes are expressed in a given cell mRNA population represents those genes expressed in a given cell (tissue specific gene expression) Thus, the mRNA of a cell gives us a snapshot of the genes which are expressed within that cell at any particular time. Central Dogma of Molecular Genetics
DNAExon1 Exon2 Exon3 IntronI IntronII D mRNA Mature mRNA D Start AAAAAAA polyA tail Stop Protein Intron splicing and polyA tailing Translation on ribosome Open Reading Frame (ORF)
Need for making cDNA The problem with mRNA is, that it cannot be maintained in stable vectors and is difficult to manipulate. DNA copy (called complementary DNA, or cDNA) of the mRNA is required before a library can be constructed.
Reverse transcriptase David Baltimore and Howard Temin first discovered the enzyme in 1970 The conversion of RNA to DNA is dependent upon the action of reverse transcriptase enzyme found in retroviruses. Reverse transcriptase is an RNA-dependent DNA polymerase that, like all other DNA polymerases, catalyses the addition of new nucleotides to a growing chain in a 5 to 3 direction. Reverse transcriptases generally have two types of enzymatic activity. 1.DNA polymerase activity: reverse transcriptase produces a DNA copy from RNA 2.RNaseH activity: RNaseH is a ribonuclease that degrades the RNA from RNA-DNA hybrids,
Construction of cDNA Isolation of mRNA – • The presence of a poly-A tail is unique to mRNA, and provides a mechanism of distinguishing and isolating mRNA from the more rRNA and tRNA molecules. • mRNA can be physically isolated by passing total RNA over a column, to which polymers of deoxythymidine (oligo-dT) are bound • RNA molecules that do not contain multiple adenine residues will be unable to adhere to such a column and will flow straight through the column. • mRNA molecules, will bind through complementary base pairing to the column And will be eluted out by changing the salt concentration.
The cloning of cDNA is initiated by mixing short (12–18 base) oligonucleotides of dT (oligo dT) with purified mRNA. The oligonucleotide will anneal to the poly-A tail of the RNA molecule. Reverse transcriptase is then added. It uses the oligo-dT as a primer to synthesize a single strand of cDNA in the presence of the four deoxynucleotide triphosphates (dNTPs). The resulting molecules will be double- stranded hybrids of one cDNA and one mRNA molecule. The primer can pair at numerous positions throughout the polyA tail and consequently will yield cDNA fragments of different lengths which may have been derived from the same mRNA molecule. First DNA strand formation
Besides priming the poly(A) sequence at the 3 end of mRNA, the′ oligo(dT) primer can also prime the poly(A) sequences present internally within mRNA. The initiation of cDNA synthesis from the oligo(dT) primed at both locations results in the generation of two truncated cDNAs from a single mRNA template. To overcome this problem, we use anchored oligo-dT primers. In addition to the 12–18 base dT sequence, anchored primers are constructed such that the extreme 3- end contains either a G, A, or C residue. Such primers will initiate DNA replication if they are paired at the extreme 5-end of the polyA tail, when the G, A, or C residue can base pair with the nucleotide immediately preceding the polyA sequence.
Second DNA strand formation In early years, a hair-pin serve as a self primer for second stand. The hair-pin would be removed from the double-stranded cDNA by treatment with S1 nuclease. However, such methods invariably resulted in the loss of sequences at the 5-end of genes. Now days the second DNA strand is synthesized in 2 ways:- Nick translation Homopolymer tailing.
• RNaseH is used to partially digest the RNA component of the RNA–DNA hybrids. • The remaining RNA is used as a primer for DNA synthesis using DNA polymerase in the presence of the four dNTPs • DNA ligase is used to seal nicks in the DNA backbone , resulting double-stranded cDNA formation. Nick translation
The RNA–DNA hybrids are treated with the enzyme terminal transferase in the presence of a single deoxynucleotide triphosphate. Terminal transferase catalyse the addition of deoxynucleotides to the 3-ends of DNA. Homopolymer tailing
The RNA of the RNA–DNA hybrids must be removed to provide a single-stranded template for new DNA synthesis. This is achieved by treating the hybrids with alkali. RNA is hydrolysed into ribonucleotides around pH 11, while DNA is resistant to hydrolysis up to about pH 13 Increasing the pH to about 12 results in the hydrolysis of the RNA, but not the DNA.
Second-strand cDNA synthesis is then initiated using an oligo- dG primer that will bind, through complementary base pairing, to the newly formed polyC sequence. Reverse transcriptase in the presence of the four dNTPs will produce the second cDNA strand.
Vector type Cloned DNA (kb) Plasmid 20 lambda phage 25 Cosmid 45 BAC (bacterial artificial chromosome) 300 YAC (yeast artificial chromosome 1000 Vector systems
1.Screening by Nucleic Acid Hybridization: The main advantage of this type of screening is that it can be applied to almost any vector system. The colonies to be screened are grown on agar plates that contain the appropriate antibiotics that allows the growth of recombinant molecules. Nitrocellulose membrane is placed on top of them and then lifted off to produce a replica version of the plate. The nylon replica is treated to lyse the bacteria and firmly attach the DNA to the sheet. Screening of recombinants
Steps:- 1. The nylon sheet is treated with alkali (e.g. 0.5 M NaOH) to initiate both bacterial cell lysis and DNA denaturation. 2. Upon neutralization, the sheet is treated with proteases (e.g. proteinase K) in order to remove the protein and leave the denatured DNA bound to the membrane. 3. The sheet is then baked at 80 C, or treated with UV light, to◦ firmly adhere the DNA to the membrane. 4. Now nitrocellulose sheet containing a denatured DNA copy of the bacterial colonies originally present on the agar plate. Hybridize the DNA on the nitrocellulose membrane with a labelled probe. It locates the colonies by radioactivity of probe, on the original dish that contain identical DNA sequences.
Probe designing Purified biochemically. Digested with the enzymes. The resulting protein fragments were subjected to amino acid sequencing All possible DNA sequences that could encode this peptide were determined Antisense DNA probe are constructed.
Immunoscreening cDNA is cloned into the expression vector under the control of the bacterial lac promoter. Recombinant λ phages are plated out onto a suitable bacterial host on agar plates. Incubated until small plaques appear. Place a nitrocellulose sheet soaked in IPTG on top of the plaques. It bind the proteins produced by the E. coli cells after lyse by the phage infection. Peel off the nitrocellulose membrane Incubate membrane with a specific antibody (against protein product of gene which we clone). Incubated membrane with a labelled secondary antibody to detect the presence of the bound primary antibody.
Screening by Function Host cell is required that either lacks a biochemical function. Those cell are screened as recombinants that are able grow. For example, Select the E. coli cells that are defective in imidazole glycerol phosphate dehydratase (essential for the biosynthesis of histidine) - unable to grow on media lacking histidine.  If the hisB defective E. coli cells are transformed with an expression library from yeast and plated onto media lacking histidine, the only recombinant cell are able to grow. The yeast HIS3 and E. coli hisB genes share little DNA sequence similarity (less than 20% ), but the encoded proteins, perform the same enzymatic function.
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