Sequencing The Polymerase Chain Reaction (PCR) and Its Applications Principles of Gene Manipulation Created almost exclusively by Jennifer Cooper America Madrigal Laleña Vellanoweth PCR The Polymerase[.]
PCR : The Polymerase Chain Reaction (PCR) and Its Applications Principles of Gene Manipulation · Created almost exclusively by Jennifer Cooper · America Madrigal · Laleña Vellanoweth 11.06.00 11.06.00 PCR: PCR and Its Applications I Definition of PCR II Requirements for PCR III PCR Process A Denaturing Stage B Annealing Stage C Extending Stage D Replication IV Some Applications of PCR A Library Screening B Forensic Identification C Making a probe D Subcloning 11.06.00 PCR What is it? The Polymerase Chain Reaction (PCR) is an in vitro method to amplify a specific region of DNA PCR is extremely sensitive, with the capability of amplifying minuscule quantities of DNA 11.06.00 11.06.00 11.06.00 PCR Why it? study DNA from fossils compare forensic samples identify remains clone subfragments that don’t have convenient restriction sites unite living members of a separated family determine tissue type for transplants amplify ds cDNA fragments from the reverse transcription products of mRNA (RT-PCR) 11.06.00 PCR REQUIREMENTS DNA sample · very small amounts (ng or sometimes less) if DNA is in good shape · may be able to use DNA from only one cell · only a few molecules must be intact samples with larger numbers of molecules can be in poor shape or degraded 11.06.00 11.06.00 PCR REQUIREMENTS (cont’d.) • • • • Two primers · flank region you are interested in · you must know the sequence of the flanking regions so you can order appropriate primers Heat stable polymerase Four dNTPs Thermocycler (standard, but optional) · changes temperature very rapidly for each cycle (denature, anneal, extend) 11.06.00 11.06.00 Programmable thermocycler 11.06.00 Same thermocycler - open Notice heating block with three samples in pink tubes 11.06.00 11.06.00 PCR METHOD There are four basic steps in PCR Denaturing Stage Annealing Stage Extending Stage Replication 11.06.00 11.06.00 PCR METHOD – DENATURING STAGE (reference: www.library.thinkquest.org/24355/data/light/details/media/polymeraseanim.html) 11.06.00 Example of STR PCR products run on an agarose gel These products are sufficiently different in length to discriminate by gel electrophoresis ... Subcloning 11.06.00 PCR What is it? The Polymerase Chain Reaction (PCR) is an in vitro method to amplify a specific region of DNA PCR is extremely sensitive, with the capability of amplifying minuscule... are interested in · you must know the sequence of the flanking regions so you can order appropriate primers Heat stable polymerase Four dNTPs Thermocycler (standard, but optional) · changes temperature... 11.06.00 PCR: PCR and Its Applications I Definition of PCR II Requirements for PCR III PCR Process A Denaturing Stage B Annealing Stage C Extending Stage D Replication IV Some Applications of PCR