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Recombinant DNA Libraries Recombinant DNA Libraries • A DNA library is a collection of DNA clones, gathered together as a source of DNA for sequencing, gene discovery, or gene function studies. – Libraries are collections of cloned DNA fragments from a particular organism contained within bacteria or viruses as the host – Libraries can be saved for relatively long periods of time and "screened" to pick out different genes of interest • Depending on the source of the DNA there are three types of rDNA libraries . Recombinant DNA Libraries 1.Genomic library, a collection of clones containing at least one copy of every DNA sequence in a genome. Genomic libraries are available for many organisms. 2.Chromosome libraries are collections of cloned fragments of individual chromosomes. 3.Complementary DNA (cDNA) libraries are cloned collections of DNA copied from a cell’s mRNA. Genomic Library • Genomic library, produced when the complete genome of a particular organism is cleaved into thousands of fragment and all the fragments are cloned by insertion into a cloning vector. • The first step in preparing a genomic library is isolation and digestion of the DNA by restriction endonucleases – This process produces fragments of DNA that include the organism's entire genome • A plasmid, BAC, YAC or bacteriophage vector is digested with the same enzyme • DNA ligase is used to ligate genomic DNA pieces and vector Genomic Libraries • In theory, all DNA fragments in the genome will be cloned into a vector • Recombinant vectors are then used to transform bacteria or yeast cells . • Each transformed bacterium or yeast cell grows into a colony, or “clone,” of identical cells, each cell bearing the same recombinant plasmid. • Consider each clone a "book“ in this "library" of DNA fragments Genomic Libraries There are three general ways to produce genomic libraries: a. Complete digestion with restriction enzyme, cleave at all relevant restriction sites. This has drawback: 1) Genes containning one or more sites for the restriction enzyme will be cloned into two or more pieces. 2) To screen the entire genome, a very large number of clones would have to be examined, because insert DNA size is relative small. b. Longer DNA fragments can be generated with mechanical sheering (e.g passage through a syringe needle) rather than restriction enzyme cutting. A disadvantage is the absence of uniform ends, require enzymatic modification before insertion into a clonning vector. Genomic Libraries c. Partial digestion with a restriction enzyme is controlled so that it cuts only some of the available sites. Ideally, this results in cloninng a population of overlapping fragments representing the entire genome . 1) Partially digested DNA molecules in a certain size range are selected by density gradient centrifugation or agarose gel electrophoresis. 2) DNA fragments with sticky ends from restriction digestion can be cloned directly 3) Genomic sequences are not equally represented in the libraries, because: a) Regions of DNA with relevant restriction sites very close together or very far apart are removed at the size selection. b) Some regions of eukaryotic DNA prevent vector replication in E. coli, and so are eliminated from the library. Genomic Libraries • One disadvantage of creating this type of library for eukaryotic genes – Is that nonprotein coding pieces of DNA called introns are cloned in addition to protein coding sequences (exons) • Because a majority of DNA in any eukaryotic organism consists of introns – Many of the clones in a genomic library will contain non-protein coding pieces of DNA • Another limitation of genomic libraries is that many organisms, including humans, have such a large genome – That searching for a gene of interest really is like searching for a needle in a haystack! cDNA Library mRNA from the tissue of interest is isolated and used for making the library However, mRNA cannot be cut directly with restriction enzymes • So it must be converted to a double-stranded DNA molecule A viral enzyme called reverse transcriptase (RT) is used to catalyze the synthesis of single-stranded DNA from the mRNA This enzyme is made by a class of viruses called retroviruses – So named because they are exceptions to the usual flow of genetic information – Instead of having a DNA genome that is used to make RNA, retroviruses have an RNA genome – After infecting host cells ,they use RT to convert RNA into DNA so that they can replicate Since this DNA has been synthesized as an exact copy of mRNA , it is called complementary DNA (cDNA) cDNA is a DNA molecule synthesied from mRNA by reverse transcriptase enzyme . cDNA Libraries The cDNA derives only from mature mRNA. Introns are not present. The poly(A) tail at the 3’ end of the mRNA is useful for: a. Isolating mRNA from cell lysates by passage over an oligo(dT) column. i. The mRNA’s poly(A) tail sticks to the poly(T) attached to the column substrate. ii. Other molecules pass through the column, but mRNAs are retained. iii. mRNAs are eluted with decreasing ionic strength buffer, resulting in significant purification. b. Priming the synthesis of cDNA, by providing a known 5’ sequence. cDNA Libraries Synthesis of cDNA involves these steps (Figure): a. A short oligo(dT) primer is used. It anneals to the mRNA’s poly(A) tail, allowing reverse transcriptase to synthesize cDNA. This creates a DNA-mRNA hybrid. b. RNase H degrades the mRNA strand, creating small RNA fragments that serve as primers. c. DNA polymerase I makes new DNA fragments, and DNA ligase connects the new DNA fragments to make a complete chain. d. The resulting cDNA is a double-stranded copy of the starting mRNA. The synthesis of complementary DNA (cDNA) from a polyadenylated mRNA A method for cloning cDNA involves : a. Introducing restriction site linkers to the ends of the cDNA by blunt end ligation. b. Digestion with the cognate restriction enzyme to create sticky ends. c. Mixing cDNA with vector DNA cut with the same restriction enzyme in the presence of DNA ligase. d. Transforming into an E. coli host for cloning. If the cDNA has sites for the same restriction enzyme used in the polylinker, the cDNA will be cloned in pieces. The problem is avoided by using polylinkers engineered with appropriate ssDNA overhangs (sticky ends) so that restriction digestion is unnecessary. The cloning of cDNA using BamHI linkers cDNA Libraries • A primary advantage of cDNA libraries over genomic libraries – Is that a cDNA library is a collection of actively expressed genes in the cells or tissue from which the mRNA was isolated – Introns are not cloned in a cDNA library, which greatly reduces the total amount of DNA that is cloned compared to a genomic library For this reason, cDNA libraries are typically preferred over genomic libraries when attempting to clone a gene of interest cDNA Libraries • One disadvantage is that cDNA libraries can be difficult to create and screen if a source tissue with an abundant amount of mRNA for the gene is not available • But polymerase chain reaction (PCR) can frequently solve this problem Chromosome libraries 1. Screening the genomic library of an organism with a large genome is laborious. Screening time can be reduced if a gene has been localized to a chromosome, by examining a library made from only that chromosome. Human, for example, have 24 different chromosome libraries (22 autosomes, X and Y). 2. Separating chromosome so they may be individually cloned is accomplished with techniques such as flow cytometry. Library Screening • Once either a genomic library or a cDNA library is created – It must be screened to identify the genes of interest • One of the most common library screening techniques is colony hybridization - In colony hybridization, bacterial colonies from the library containing recombinant DNA are grown on an agar plate • Hybridization is a general technique for detecting a specific gene or mRNA, by allowing a labeled probe with the complementary sequence to bind to the target molecule. Probes • Probes are either cloned or synthetic fragment of single strande nucleic acid ( usually DNA but may be RNA ) used in either DNA:DNA or DNA:RNA hyperdization ) • Probes are usually 20 to 30 bases long and can be labeled with radioactive tags or fluorescent tags. • Probe is capable of hyperdizing with a DNA fragments that carry the complementary sequence . • Types of probes : - Cloned DNA probes . - cDNA probes . - Synthetic oligonucleotide probes . - What is the difference between cloned probe and cDNA probe ? Heterologous Probes It is possible to identify specific genes in a genomic library using cloned equivalent genes (heterologous probes) from other organisms, especially if the gene is highly conserved or the species are closely related.(many gene sequences in rats and mice are similar to those found in human genes ) Synthetic Oligonucleotide Probes 1. Synthetic oligonucleotides are useful in probing libraries, sequence data are available for part of the gene of interest. Knowledge of substitutions produced by mutation also aids probe selection. Sequences for many genes are available in GenBank. 2. Using the universal genetic code, the amino acid sequence is used to design a DNA oligonucleotide probe. Degeneracy in the genetic code means that a mixture of oligonucleotides must be prepared, each of which encodes the target protein. 3. The library is probed with the oligonucleotide mixture to detect the gene of interest. Library Screening Clones detected by the probe are characterized Screening a cDNA library One way to select a cDNA clone from the library is to detect a protein product it is producing (Figure). For protein to be produced, an expression vector is needed, in which the cloned cDNA is inserted between a promoter and a transcription terminator. Labeled antibodies are used to detect the specific protein in a host colony. An array of colonies is transferred to a membrane fiter, cells are lysed and their proteins bind to the filter, which is incubated with the relevant antibody. Radioactively labeled antibody bound to colonies is detected by an autoradiogram, in which the dry fiter is placed on X ray film in the dark for a number of hours. Colonies with antibody bound will be visible as dark spots on the film. Once identified, the cDNA can be used . Screening for specific cDNA plasmids in a cDNA Library by using an antibody probe The antibody binds to a specific Site on a protein that is made via The inserted foreign DNA. This Is a Western Blot since it uses antibody To detect a protein.