STANDARD CLONING PROCEDURES. Shotgun cloning (using a plasmid vector and E coli as a host).

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1 STANDARD CLONING PROCEDURES Shotgun cloning (using a plasmid vector and E coli as a host). 1) Digest donor DNA and plasmid DNA with the same restriction endonuclease 2) Mix the fragments together and treat with DNA ligase 3) Transform the plasmids into competent E. coli cells electroporation (an electric shock creates pores) Osmotic shock of chilled cells makes membranes permeable 4) Plate the treated cells on medium containing an antibiotic so that only cells with a plasmid containing a resistance gene can grow 5) If a puc plasmid was used, include X-GAL and and IPTG (an artificial inducer for the lac operon) in the medium so that colonies that only contain intact vector will turn blue, while those with a foreign DNA inserted will remain white. cdna cloning (advantageous for cloning eukaryotic genes into E. coli ) 1) Isolate mrna from target tissue; use oligodt beads or columns to pull messages out of solution by their tails! 2) Add oligo-dt to serve as a primer for Reverse Transcriptase (this creates a DNA complimentary strand hybridized to the message) 3) Add RNase H, DNA Polymerase I and DNA ligase. (These enzymes will replace the RNA strand with DNA.) 4) Add tails with TdT or add linkers by blunt end ligation to match prepared tails on the vector

2 5) Mix prepared vector with cdna copies and proceed as in step 3 above. Bacteria cannot remove introns from eukaryotic genes; therefore, cdna clones but not shotgun clones can make the correct polypeptide. IDENTIFYING CLONED GENES Hybridize to known clone from another species Compare base sequence to known genes in GenBank Sanger sequencing; Fragments cloned into many vectors can be sequenced by using a DNA polymerase (often the Klenow fragment of E. coli DNA Polymerase I or a modified version of T7 DNA polymerase) to extend a primer complementary to a flanking site in the vector. M13 forward and reverse primer sites flank the MCS of many vectors and the primers are readily available commercially. The procedure produces a set of variable length fragments because: -dideoxy-nucleotides (ddntp) cause the DNA polymerase to stop if inserted into a growing chain - a different dideoxy base is present in each of four reactions, along with all 4 dntps (or each base can be labeled with a different colored fluorescent dye) - a radioactive dntp is added for detection, or a different flouresent tag may be added to each ddntp - < 1% chance to stop at each base - the fragments produced are separated by polyacrylamide gel electrophoresis -the length of each fragment reveals the last base added

3 Matching sequences in GenBank can be identified by a BLAST search Complementation: The gene is expressed in a different organism or replaces a function missing in a mutant host Express the gene (ie, transcribe and translate mrna) and detect the protein with an antibody made against purified protein, or show that the correct enzyme is made by showing enzymatic activity (some polypeptides require modifications or co-factors that may not be present in E. coli to function correctly.) 2 Techniques: DNA PROBES Southern Blots Northern Blots DNA/DNA hybridization detect RNA with a DNA probe Applications: Mapping genes -RFLPs segregate as codominant alleles - in situ hybridization on chromosome Estimate gene copy number or quantify mrna Species-specific diagnostic probes Population analysis/forensics -Hypervariable alleles -Fingerprinting probes, repeated sequences

4 Prenatal detection and heterozygote screening amniocentesis fetal cells collected at 13 wks chorionic villus sampling (CVS) samples collected at 8 weeks Defects detectable: chromosome aberrations (Down's etc) enzymes or gene products (Tay-Sachs, etc) DNA changes (sickle cell etc) -RFLPs -allele-specific probes 3 Technique: DNA AMPLIFICATION - PCR (Polymerase Chain Reaction) Add short primers flanking desired sequence Heat DNA to melt into single strands Cool, extend primers with Taq DNA polymerase cycle temperature 20-40X in <4 hr Applications: Cloning and sequencing specific sequences

5 Forensics; variable regions from a single DNA -ITS of rdna genes -HLA-D4 alleles -Variable mtdna sequences Diagnosis; amplify species-specific sequences Prenatal detection and heterozygote screening -Sickle cell; amplify, then cut? answer in less than a day -SSCP-PCR detects single base differences -Y specific sequences 4 GENE THERAPY/TRANSGENIC AND EXOTIC ORGANISMS Gene Delivery Mechanisms transformation of "returnable" cells virus vectors

6 microinjection microparticle bombardment - the "gene gun" a natural system for plants - the TI plasmid 5

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