DNA Libraries and Screening for Recombinants PDF

DNA Libraries and Screening for Recombinants NURRIZA AB LATIF (PhD) Dept. of Biosciences, Faculty of Science, UTM  DNA 01 LIBRARIES What is DNA library? A set of DNA clones representing either the entire genome of an...

DNA Libraries and Screening for Recombinants NURRIZA AB LATIF (PhD) Dept. of Biosciences, Faculty of Science, UTM  DNA 01 LIBRARIES What is DNA library? A set of DNA clones representing either the entire genome of an organism or a set of genes that are expressed in a cell type. GENE LIBRARIES Sets of cloned DNA fragments that together represent the genes of a particular organism. Any particular gene may represent a tiny, tiny fraction of the DNA in a given cell. Can't isolate it directly – it is impractical to attempt to recover such rare sequences directly from isolated nuclear DNA because of the overwhelming amount of extraneous DNA sequences.  The trick – to find the fragment or fragments in the library that contain the desired gene. A genomic library is prepared by isolating total DNA from the organism, digesting it into fragments of suitable size, and cloning the fragments into an appropriate vector. Such a collection from which the desired clone is withdrawn is called a gene bank or gene library. This approach is also called shotgun cloning because the strategy has no way of targeting a particular gene but instead seeks to clone all the genes of the organism at one time.  The intent is that at least one recombinant clone will contain at least part of the gene of interest. Usually, the isolated DNA is only partially digested by the chosen restriction endonuclease so that not every restriction site is cleaved in every DNA molecule. Even if the gene of interest contains a susceptible restriction site, some intact genes might still be found in the digest. http://www.sumanasinc.com/webcontent/animations/content/dnalibrary.html Generation of overlapping fragments in partial digest Fragments between 10-20 kb chosen  Gene/genomic library - traditionally made using phage λ as vector because of λ is able to incorporate large pieces of DNA. Other vectors (YACs, yeast artificial chromosomes ~1000kb; BACs, bacterial artificial chromosomes ~500kb) are now frequently used. Genomic libraries are made by cutting the target DNA with restriction enzymes and ligating to an appropriate vector.  DNA library gDNA library cDNA library  CONSTRUCTION 02 OF GENOMIC LIBRARY Steps in genomic library construction 1. Isolate Genomic DNA Obtain the DNA: Extract genomic DNA from the organism of interest. Ensure Quality: Use methods like phenol-chloroform extraction and ethanol precipitation to ensure high-quality DNA. Quantify and check purity using a spectrophotometer or agarose gel electrophoresis. 2. Fragmentation Restriction Enzymes: Use one or more restriction enzymes to cut the genomic DNA into manageable fragments. Mechanical Shearing: Alternatively, use mechanical methods like sonication to randomly break the DNA. Size Selection: Run the fragmented DNA on an agarose gel and select the desired size range using gel extraction methods. 3. Prepare the Vector Choose a Suitable Vector: Depending on the size of the fragments, use: Plasmids (for smaller fragments, 1–10 kb) Lambda phage vectors (10–20 kb) BACs (bacterial artificial chromosomes, 100–300 kb) YACs (yeast artificial chromosomes, >300 kb) Digest the Vector: Use compatible restriction enzymes to open the vector at the appropriate site. Dephosphorylation: Treat the vector with alkaline phosphatase to prevent self-ligation. 4. Ligate DNA Fragments into the Vector Mix the genomic DNA fragments with the prepared vector in the presence of DNA ligase and ATP. The ligase joins the DNA fragments to the vector, forming recombinant DNA. 5. Transform Host Cells Introduce the Recombinant DNA: Transform the ligated DNA into a host organism (e.g., E. coli) using methods like: Heat shock (for plasmids) Electroporation (for BACs and YACs) In vitro packaging systems (for lambda phage vectors). 6. Plate the Transformants Spread the transformed cells on selective agar plates containing antibiotics or other markers to select for successful transformants. 7. Screen the Library Colony Hybridization: Use labeled probes (radioactive or fluorescent) to identify colonies containing specific DNA sequences. PCR-Based Screening: Amplify target sequences to identify the desired clones. 8. Store and Maintain the Library Store the genomic library as glycerol stocks at –80°C or as DNA libraries in appropriate buffers for long-term use.  Applications of Genomic Library Identifying genes 01. 02. Sequencing entire and regulatory genomes. elements. Producing Studying genetic 03. 04. recombinant proteins disorders and or transgenic mutations. organisms. 03 cDNA CLONING What is cDNA library? A collection of DNA clones representing the expressed gene of an organism in a cell. cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an organism. cDNA library In eukaryotic cells : DNA contain intron and exon However, the mRNA produce from the transcription process lack of intron Intron and Exon in Eukaryotic Cells exon exon exon promoter intron intron DNA 3’ 5’ start codon stop codon Transcription 5’ 3’ mRNA Processing cap AAA A poly A Splicing tail Intron deleted AAA A mature mRNA To cytoplasm  Splicing - removal of RNA introns in nucleus. That’s why cDNA derived from mRNA lacks introns. RNA splicing which is a post- transcriptional process in eukaryotes do not occur in bacteria. Construction of cDNA library STEP 1 – Isolation of mRNA (most critical step) ***All components used should be super clean and free of chromosomal DNA, RNases and phenolics. STEP 2 – Synthesis of 1st strand of DNA STEP 3 – Synthesis of 2nd strand of DNA  In the cell Complementary DNA is DNA made in vitro using mRNA as a template and the enzyme reverse transcriptase. In test tube Key molecules in cDNA construction: Reverse transcriptase (RTase) Ø Synthesize 1st strand of cDNA using mRNA as template oligo dT primers Ø Essential in mRNA purification DNA Polymerase Ø Synthesize 2nd strand of cDNA using 1st strand cDNA as template S1 nuclease Ø Digest hairpin loop Total RNA and mRNA Isolation Isolation of full length and undegraded mRNA is crucial to ensure success of the cDNA to be synthesized. To prevent RNAse contamination and to eliminate RNAses, several steps have to be taken: 1. All plasticware (tips, Eppendorf tubes, and dd water) has to be treated with DEPC (diethylpyrocarbonate) overnight and autoclaved to remove the DEPC. 2. Glassware, ceramics (mortar and pestle, spatula) have to be baked at 200oC. 3. The grinding of the cell mass or tissue in liquid nitrogen helps to retard RNAses.  cDNA library construction RNA extraction Full length and intact RNA is crucial for cDNA library construction Precaution before RNA extraction.. Eliminate all source of RNAse: All plastic wares need to be treated with diethylpyrocarbonate (DEPC) Glasswares and ceramics need to be baked at 200oC Grinding sample in LN can inhibit the RNAse activity Need to change glove frequently to avoid RNAse contamination After you have your RNA …. 2. Purify the mRNA DNA need to be eliminated. Remove rRNA and tRNA How to isolate the pure mRNA? Ø Isolate the mRNA using matrix column containing Oligo (dT) Purification of mRNA using Oligo dT column Oligo dT column: How it works? RT add the dNTP at 3’-OH Formation of loop DNA Pol add the dNTP at 3’- OH to synthesize the 2nd strand of cDNA  Prior to the vector ligation, linker, adapter or homopolymer can be added to the cDNA If a bacterial host is used, Ø transform the recombinant DNA into cell Ø plate cells on the selection media  After you have your cDNA library what you going to do? After you have your cDNA library what you going to do? Sequence the whole library to determine the function of each expressed gene Detect the specific gene of interest using probe Application of cDNA library Storage of reduced amount of information due to the removal of non-coding regions. cDNA can be directly expressed in prokaryotic organisms. cDNA libraries are useful in reverse genetics where the additional genomic information is of less use. cDNA library is useful for isolating gene that codes for particular mRNA. cDNA Library vs. Genomic DNA Library cDNA library lacks the non-coding and regulatory elements found in genomic DNA. Genomic DNA libraries provide more detailed information about the organism, but are more resource-intensive to generate and maintain. 03 SCREENING OF A LIBRARY One of the most difficult task in gene cloning is finding the right shelf in a genomic library – identifying the bacterial or phage clone containing a desired gene.  The process of identifying the clone or clones that have the gene of interest out of several hundred thousand clones. Using probes to identify the cloned sequence. A probe = a piece of DNA or RNA used to detect specific nucleic acid sequences by hybridization (binding of two nucleic acid chains by base pairing).  They can be radioactively labelled so that the hybridized nucleic acid can be identified by autoradiography. The size of probes ranges from a few nucleotides to hundreds of kb. Originally they may be double-stranded, but the working probes must be single-stranded. Short probes (oligonucleotide probes) can be made by chemical synthesis.  Screening Methods for DNA Library Hybridization- Based Screening Screening PCR-Based methods Screening Immunological Screening Hybridization with a DNA probe Antibody Screening – Expression Screening Indirect identification of translation products - antibody screening. If the purified product of a given gene is available in sufficient quantity, antibodies specific for that product can be raised. These can then be used to identify clones expressing that protein and thus containing the gene encoding it. Ø The procedure is similar to that used for hybridization screening.  Recombinant plaques or colonies are transferred to nitrocellulose or nylon filters and then exposed to the specific antibody. The antibody recognizes and binds to any region of the filter in which the target protein is present. Unbound antibody is then carefully washed from the filter and any specifically retained material is identified with the use of a second antibody. The second antibody is specific for the IgG of the species in which the first antibody was raised and is tagged with an enzymatic or radioactive label.  Reaction of protein with its specific antibody http://www.cellsignal.com/products/images/wb.jpg Construct a λ library designed to express the protein encoded by the cDNA Transfer the induced proteins made by the phage to a filter Screen the plaques with an antibody directed against your protein of interest Probe Labeling: Method I Random hexamer and fill-in reaction with Klenow fragment; Taq polymerase Probe Labeling: Method II End label with T7 polynucleotide kinase Probe Labeling: Method III Colony Hybridization The procedure of screening a plasmid library for specific clones using a probe. Steps: 1. Spread colonies from the cDNA library on to nutrient plates. 2. A replica of the colonies on the plate is made by pressing a nitrocellulose filter on to the surface. 3. Then the filter is passed through solutions to lyse the bacteria, denature ds DNA to ss DNA and bind to the filter. 4. The filter is incubated with the radiolabelled ss DNA probe. 5. If DNA sequence of the any of the cloned DNA on the filter finds the complementary to the probe, it will hybridize. 6. The filter is overlaid with a piece of X-ray film and when developed, the hybridized molecules will be shown as dark spots. 7. These represent colonies on the plate containing the cloned gene of interest. Screening of Recombinant Plasmid Library Screening of Recombinant Phage Library Screening a Recombinant Library for a Clone Expressed in a Specific Tissue Note: each phage clone represents a copy (cDNA) of an mRNA from the cells used to make the cDNA library Thank you