STANDARD CLONING PROCEDURES. 1) Digest donor DNA and vector (assuming a plasmid) DNA with the same restriction
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1 STANDARD CLONING PROCEDURES Shotgun cloning: (steps using a plasmid vector and E coli as a host). 1) Digest donor DNA and vector (assuming a 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 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.
2 IDENTIFYING CLONED GENES Express the gene in a host lacking the sought-for function (complementation) Hybridize to a clone of the same gene already cloned from another species (bacterial colonies in a plasmid library can be replicated onto a membrane, lysed and hybridized as below (colony hybridization)) (the clones in a virus library can also be detected by transfer to a membrane (plaque hybridization)) Show that the expressed protein a) catalyzes the desired reaction, (In some cases, modifications or co-factors that are required for activity may not be present in E. coli to function correctly) b) is bound by antibody made against the actual protein, c) encodes the expected amino acid sequence Compare base sequence to known genes in GenBank Sequencing cloned DNA 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 phage 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. Sanger di-deoxy sequencing Four DNA polymerase reactions (or one reaction using different fluorescent dyes for each base) are made, each including all 4 dntps plus a small amount of a di-deoxy ribose of one of the nucleotides Insertion of a di-deoxy-nucleotide (ddntp) ends the DNA polymerase reaction (< 1% chance to stop at each position during extension)
3 A radioactive dntp is added for detection, or a different fluorescent tag may be added to each ddntp The fragments produced in each reaction are separated by polyacrylamide gel electrophoresis -the length of each fragment reveals the last base added radio-label example: Florescent label example DNA PROBES Southern Blots: DNA/DNA hybridization DNA fragments are separated by electrophoresis in agarose gels small fragments migrate rapidly large fragments remain near the well The DNA fragments are denatured (made single stranded) and "blotted" onto Nylon membranes Fragments are locked onto the membrane by UV cross-linking or by simply drying the membrane Probes (cloned DNA inserts) are labeled using DNA polymerase, random primers, and a radioactive or modified/tagged base Probes are boiled, added in a hybridization solution to the membrane and allowed to "anneal" at a selected temperature (usually about 55 ) overnight Nonhybridized probes are washed off the membrane and it is exposed to photographic film (assuming a radioactive or light emitting label used) Dark spots seen on developing the film locate the target DNA with a sequence complementary to the probe For an example, see
4 Examples of Applications: Mapping genes -RFLPs segregate as codominant alleles - in situhybridization; hybridize the probe to a chromosome Estimate gene copy number Species-specific diagnostic probes Population analysis/forensics Hypervariable alleles Fingerprinting probes, repeated sequences Northern blots and hybridization Northerns refer to detecting RNA using a DNA probe Can be used to quantify amount of mrna Can be used in situ to detect tissue-specific gene expression Western hybridization Detecting proteins using labeled antibodies PCR AMPLIFICATION (Polymerase Chain Reaction) Technique: Add short primers flanking the desired sequence Heat DNA to 95 C to melt target DNA into single strands Cool to to allow primers to anneal Raise to 72 extend primers with Taq DNA polymerase (Taq polymerase comes from Thermus aquataus, a hot springs bacterium so can withstand high temperatures) Cycle between melting, annealing and extension temperatures times (modern thermal cyclers can heat and cool in a few seconds) Applications: Cloning and sequencing of DNA sequences ("Reverse Genetics")
5 If part of the amino acid sequence for a protein is known, forward and reverse primers can be designed using the genetic code -Chose amino acids with as few codons as possible -Degenerate primers (made with a mix of 2-4 bases at a specific position) can be used; one will be correct) -the amplified fragment can be used as a probe to detect cdnas or can be extended to full length cdnas using 5' and 3' RACE (Rapid amplification of cdna ends) Regions of high sequence homology can be identified from searches of the same enzyme or DNA sequences available in GenBank, and used to design primers (degenerate if needed) Diagnostics: forensics, species identification, prenatal diagnostics, heterozygote identification -variable sequences between conserved sequences are amplified Amplified products sized by electrophoresis Amplified products can be sequenced -Examples of variable regions used include: -ITS of rdna genes -HLA-D4 alleles -Variable mtdna sequences -SSRs (simple sequence repeats, such as trinucleotide repeats GENE THERAPY/TRANSGENIC AND EXOTIC ORGANISMS Gene Delivery Mechanisms transformation of "returnable" cells virus vectors microinjection microparticle bombardment - the "gene gun"
6 a natural system for plants - the TI plasmid from Agrobacterium tumefaciens, (TI stands for tumor inducing, a symptom of plants infected by the "crown gall" bacteria) -the TI plasmid is transferred into the host chromosome -normal TI genes are replaced by a cloned gene of interest and a selectable marker, such as hygromycin resistance -infected callus cells are grown on hygromycin before regenerating whole plants that carry the transgene
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