Lec 4 PDF - DNA Sequencing and Polymerase Chain Reaction

6-Polymerase Chain Reaction (PCR) It is a test tube (In vitro) method for DNA multiplication (In vitro gene cloning ) As with any DNA polymerase reaction it requires: 1-DNA template: it is provided by the DNA sample to be amplified 2-Free 3′-OH (primer): to get the polymerase started. It is provided...

6-Polymerase Chain Reaction (PCR) It is a test tube (In vitro) method for DNA multiplication (In vitro gene cloning ) As with any DNA polymerase reaction it requires: 1-DNA template: it is provided by the DNA sample to be amplified 2-Free 3′-OH (primer): to get the polymerase started. It is provided by site-specific oligonucleotide. The primers are complementary to each of the ends of the sequence that is to be amplified. 3-Substrates: deoxynucleotide triphosphates (dNTPs). Present in excess. 4-Thermostable DNA polymerase: Taq. Polymerase 5-Optimum temperature: provided by thermal cycler 6-Optimum pH (enzyme buffer). 7-Reaction volume of H2O: must be deionized any ions will ppt. DNA 1) Denaturation: the target sequence of DNA is heated to denature the template strands and render the DNA single-stranded. 2) Annealing: The DNA is then cooled to allow the primers to anneal, that is, to bind the appropriate complementary strand. The temperature for this step varies depending on the size of the primer, the GC content, and its homology to the target DNA. 3) Primer extension: In the presence of Mg2+, DNA polymerase extends the primers on both strands from 5′ to 3′ by its polymerase activity. Primer extension is performed at a temperature optimal for the particular polymerase that is used. Currently, the most popular enzyme for this step is Taq polymerase, the DNA polymerase from the thermophilic (heat-loving) bacteria Thermus aquaticus. This organism lives in hot springs that can be near boiling and thus requires a thermostable polymerase. These three steps are repeated from 28 to 35 times. With each cycle, more and more fragments are generated with just the region between the primers amplified. These accumulate exponentially. The contribution of strands with extension beyond the target sequence becomes negligible since these accumulate in a linear manner. After 25 cycles in an automated thermocycler machine, there is a 225 amplification of the target sequence. PCR products can be visualized on a gel stained with nucleic acid-specific fluorescent compounds such as ethidium bromide or SYBR green. Radio-labeled DNA by using radio-labeled dNTPs and examination under X-ray. When Kary Mullis first developed the PCR method in 1985, his experiments used E. coli DNA polymerase. Because E. coli DNA polymerase is heat-sensitive, its activity was destroyed during the denaturation step at 95°C. Therefore, a new aliquot of the enzyme had to be added in each cycle. Using of the DNA polymerase from T. aquaticus made the reaction much simpler. In his first experiments, Mullis had to move the reaction manually between the different temperatures. Fortunately, this procedure has been automated by the development of thermal cyclers. These instruments have the capability of rapidly switching between the different temperatures that are required for the PCR reaction. Thus the reactions can be set up and placed in the thermal cycler, and the researcher can return several hours later (or the next morning) to obtain the products.  7-DNA sequencing  DNA sequencing is used to provide the ultimate characterization once a gene has been cloned or amplified by PCR.  Uses of DNA sequencing: 1) Identification of genes, 2) Determine the sequence of promoters and other regulatory DNA elements that control expression, 3) Confirm the DNA sequence of cDNA and other DNA synthesized in vitro 4) Help to deduce the amino acid sequence of a gene or cDNA from the DNA sequence.  There are two types of DNA sequencing: a) Manual DNA sequencing by the Sanger “dideoxy ” DNA method b) Automated DNA sequencing a) Manual DNA sequencing  The most widely used method for manual DNA sequencing is the Sanger or “dideoxy” method, which is, in essence, a DNA synthesis reaction. 1) ssDNA is mixed with a radioactively labeled primer to provide the 3′-OH required for DNA polymerase to initiate DNA synthesis. 2) The primer is usually complementary to a region of the vector just outside the multiple cloning site. 3) The sample is then split into four aliquots, each containing DNA polymerase, four dNTPs (at high concentration), and a low concentration of a replication terminator (it is dideoxynucleoside triphosphates (ddNTPs) that are missing the 3′-OH. Because they lack the 3′-OH, they cannot form a phosphodiester bond with another nucleotide. 4 ) Each reaction proceeds until a replication-terminating nucleotide is added, and each of the four sequencing reactions produces a series of ssDNA molecules, each one base longer than the last. 5 ) The sequencing mixtures are loaded into separate lanes of a denaturing PAGE to separate the DNA fragments. 6 ) Autoradiography is used to detect a ladder of radioactive bands. 7 ) The radioactive label (primer) is at the 5′ end of each newly synthesized DNA molecule. Thus, the smallest fragment at the bottom of the gel represents the 5′ end of the DNA. 8) Reading the sequence of bases from the bottom up (5′→3′) gives the sequence of the DNA molecule synthesized in the sequencing reaction. 9) The sequence of the original strand of DNA is complementary to the sequence read from the gel (3′ → 5′)  Automated DNA sequencing In this new sequencing technology, radioactive markers are replaced with fluorescent ones. Each ddNTP terminator is tagged with a different color of fluorophore: red, green, blue, or yellow. Thus, instead of having to run four separate sequencing reactions, the reactions can be combined into one tube. The first automated sequencer made use of a polyacrylamide gel to resolve the samples, a laser to excite the dye molecules as they reached a detector near the end of the gel, and a computer to read the results as a DNA sequence. In this system each automated sequencer was able to produce 4800 bases of sequence per day. The current automated systems replace the old-style gel with arrays of tiny capillaries, each of which acts as a “lane.”