Recitation CHAPTER 9 DNA Technologies
DNA Cloning: General Scheme A cloning vector and eukaryotic chromosomes are separately cleaved with the same restriction endonuclease. (A single chromosome is shown here for simplicity.) The fragments to be cloned are then ligated to the cloning vector. The resulting recombinant DNA (only one recombinant vector is shown here) is introduced into a host cell, where it can be propagated (cloned). Note that this drawing is not to scale: the size of the E. coli chromosome relative to that of a typical cloning vector (such as a plasmid) is much greater than depicted here.
Cleavage of DNA molecules by restriction endonucleases. Restriction endonucleases recognize and cleave only specific sequences, leaving either (a) sticky ends (with protruding single strands) or (b) blunt ends. Fragments can be ligated to other DNAs, such as the cleaved cloning vector (a plasmid) shown here. This reaction is facilitated by the annealing of complementary sticky ends. Ligation is less efficient for DNA fragments with blunt ends than for those with complementary sticky ends, and DNA fragments with different (noncomplementary) sticky ends generally are not ligated. (c) A synthetic DNA fragment with recognition sequences for several restriction endonucleases can be inserted into a plasmid that has been cleaved by a restriction endonuclease. The insert is called a linker; an insert with multiple restriction sites is called a polylinker.
Typical Expression Vector DNA sequences in a typical E. coli expression vector. The gene to be expressed is inserted into one of the restriction sites in the polylinker, near the promoter (P), with the end of the gene encoding the amino terminus of the protein positioned closest to the promoter. The promoter allows efficient transcription of the inserted gene, and the transcription-termination sequence sometimes improves the amount and stability of the mrna produced. The operator (O) permits regulation by a repressor that binds to it. The ribosomebinding site provides sequence signals for the efficient translation of the mrna derived from the gene. The selectable marker allows the selection of cells containing the recombinant DNA.
Enzyme that covalently joins two DNA fragments Normally function in DNA repair Human DNA ligase uses ATP Bacterial DNA ligase uses NAD DNA Ligase
Expression of Cloned Genes We want to study the protein product of the gene Special plasmids, called expression vectors, contain sequences that allow transcription of the inserted gene Expression vectors differ from cloning vectors by having: Promoter sequences Operator sequences Code for ribosome binding site Transcription termination sequences
Specific expression vector that I have used.
Unique expression vectorthat I have used, allows the expression of a hetrodimeric protien.
Purification of Recombinant Genes Purification of natural proteins is difficult Recombinant proteins can be tagged for purification The tag binds to the affinity resin, binding the protein of interest to a purification column
Polymerase Chain Reaction (PCR) Used to amplify DNA in the test tube Can amplify regions of interest (genes) within linear DNA Can amplify complete circular plasmids Mix together Target DNA Primers (oligonucleotides complementary to target) Nucleotides: datp, dctp, dgtp, dttp Thermostable DNA polymerase Place the mixture into thermocycler Melt DNA at about 95 C Cool separated strands to about 50 60 C Primers anneal to the target Polymerase extends primers in 5 3 direction After a round of elongation is done, repeat steps
General Steps of PCR Amplification of a DNA segment by the polymerase chain reaction (PCR). (a) The PCR procedure has three steps. DNA strands are 1 separated by heating, then 2 annealed to an excess of short synthetic DNA primers (orange) that flank the region to be amplified (dark blue); 3 new DNA is synthesized by polymerization catalyzed by DNA polymerase. The three steps are repeated for 25 or 30 cycles. The thermostable Taq DNA polymerase (from Thermus aquaticus, a bacterial species that grows in hot springs) is not denatured by the heating steps.
Construction of cdna mrna can be extracted from eukaryotic cells All mrna molecules have poly-a tail Helps in purification of mrna Serves as a universal template DNA strand can be synthesized using mrna as a template This is catalyzed by the reverse transcriptase The end result is a hybrid where the DNA strand is complementary to the mrna The hybrid can be converted to duplex DNA, known as cdna
Construction of cdna Building a cdna library from mrna. A cell s total mrna content includes transcripts from thousands of genes, and the cdnas generated from this mrna are correspondingly heterogeneous. Reverse transcriptase can synthesize DNA on an RNA or a DNA template. To prime the synthesis of a second DNA strand, oligonucleotides of known sequence are ligated to the 3 end of the first strand, and the double-stranded cdna so produced is cloned into a plasmid.
Site-Directed Mutagenesis
Site-Directed Mutagenesis Understanding the function of proteins often requires that a specific amino acid residue be mutated To mutate an amino acid, change the nucleotide(s) in the coding DNA and express the mutated gene Site-directed mutagenesis usually relies on chemically synthesized mutated primers that are incorporated into newly synthesized DNA Mutated plasmids are always sequenced to confirm the desired (and only the desired) mutation is present