1 PCR: identification, amplification, or cloning of DNA through DNA synthesis DNA synthesis, whether PCR or DNA replication in a cell, is carried out by DNA polymerase. DNA polymerases have three requirements: –Only work in a 5’ to 3’ direction –Require a template: DNA to copy. –Require a primer: a free 3’ OH end to add to. PCR uses Taq polymerase, a heat stable polymerase from the thermophilic bacterium Thermus aquaticus.

2 Facts about PCR PCR requires sequence information –Primers on either side of area of interest must be complementary, thus you must know sequence. Works best on fairly small fragments of DNA Theoretically, can work on a single DNA molecule. –This is where PCR really shines. –Is a method for amplification: make enough DNA to see a band on a gel; substitute for traditional cloning.

3 PCR Template DNA Primers dNTPs Taq polymerase 1.Denature (high heat) 2.Anneal primers 3.Synthesize 4.Repeat

4 Some PCR details Primers: nucleotides long, typically Geometric increase in DNA: from 1 molecule, 34 million in 25 cycles bp limit in size Most reliable when starting with at least DNA molecules. mov.htmlhttp://faculty.plattsburgh.edu/donald.slish/PCR mov.html MANY animations online.

5 Using reverse transcriptase Enzyme that makes DNA from RNA –Product of retroviruses –Can be amplified by using PCR Library of expressed DNA using cDNA –If expressed, then can be collected as mRNA –Reverse transcriptase can make DNA from RNA template

6 Action of reverse transcriptase Poly-T synthetic oligonucleotide acts as primer, mRNA is the template. DNA-RNA hybrid is denatured, RNA is digested away, and DNA polymerase makes a ds DNA of the ss DNA. This piece of DNA, representing an active gene, can now be cloned.

7 Finding a clone in a mixture Finding a clone anywhere involves using a probe. –A probe is a nucleic acid that is sufficiently complementary to the DNA of interest that it will base-pair with it. –The probe must be labeled or else the binding will be invisible (and thus worthless) –Labeling is usually with radioactive isotopes or fluorescent compounds Detection is with x-ray film

8 DNA hybridization Use of a probe results in a ds molecule where the strands are from different sources: hybrid. Heat (or alkali) required to denature DNA (ss) Done at high salt –Na + stops PO 4 - from repelling each other Done at elevated temperature –Incorrect (weak) matches between strands come apart, allow correct ones to form. Complementary strands “zip up” once they find each other. If you have primers, you can use PCR instead to find your DNA: the one that gets amplified.

9 Colony (in situ) hybridization For finding e.g. bacterial colonies that contain your clone. Cells in colonies on a plate are lysed, the DNA is denatured and transferred to a membrane. Membrane is mixed with probe (complementary and labeled) –Loose probe is washed away –Membrane covered with x-ray film –Colony with clone of interest makes spot on film

10 Southern Blot

11 DNA sequencing DNA sequencing is a method that reveals the sequence of nucleotides in a stretch of DNA. Sequencing requires DNA synthesis; you actually determine the sequence of a complementary strand. A template and primer are mixed with a polymerase, dNTPs, and a smaller proportion of ddNTPs

12 DNA sequencing-2 ddNTPs (Dideoxy NTPs) are missing an -OH group at the 3’ position. Remember that this is where the next nucleotide must attach during DNA synthesis. There are many DNA molecules in a test tube, all being copied at the same time. If a ddNTP is inserted into the growing DNA strand, synthesis of that molecule STOPS.

13 DNA sequencing-3 ddNTPs are inserted at random (that is, when G is called for, sometimes the enzyme will grab ddGTP) This process results in a collection of DNA molecules of different lengths that can be separated on a gel. Since all 4 ddNTPs are present, growth of the DNA can stop after any base, producing a large number of DNA molecules that differ in size by 1 base. Each ddNTP is fluorescently labeled with a different color. The whole mixture is separated in a run-off gel, and the different colors marking which base terminated the reaction can be detected.

14 DNA sequencing-4 This picture shows a few of the different sized molecules that can result. Notice that each ends with a particular ddNTP; in modern practice, this will have a color associated with it and thus be identified. derived from opbs.okstate.edu/.../ img015.jpg

15 DNA sequencing gel carnegieinstitution.org/.../ sequencinggel.html Inexpensive, older method: Four reaction tubes, each containing a different ddNTP: ddATP, ddCTP, etc. Contents of each tube run in a different lane, stained to see the bands. Gel read from bottom up.

16 DNA sequencing w/ fluorescent labels ddCTP = blue ddATP = green ddTTP = red ddGTP = grey medlib.med.utah.edu/.../biochem/ Formosa/menu.html