Professional Documents
Culture Documents
PCR Process
Step 1: Set up the reaction tube (add all ingredients): Template DNA 2 Primers (at opposite ends of the target sequence) Nucleotides DNA Polymerase
Step 2: Denaturation - increase the temperature to 95oC to separate the double-stranded DNA molecule into 2 single-stranded molecules Step 3: Anneal - decrease temperature to 50oC to allow primers to bind to the single stranded DNA molecules Step 4: Polymerization - raise the temperature to 72oC (temperature at which Taq Polymerase functions best) and allow Taq Polymerase to extend/replicate the DNA strands (doubles the number of copies of the original DNA) the polymerase stops when it runs out of template (the strand ends) Step 5: Cycling - repeat steps 2-4 approximately 30 times (there are more templates for replication after each cycle is completed short templates rapidly outnumber the long templates resulting from and including the original DNA)
Important Points
What is so special about the third cycle of PCR? the first complete short double-stranded segments of the targeted sequence emerge How quickly do the shorter fragments outnumber the long ones? by the fifth cycle
HOW CAN PCR BE USED FOR GENETIC ENGINEERING? THE PRODUCT OF PCR CAN BE INSERTED INTO A PLASMID AND CLONED . POINT MUTATIONS CAN BE MADE DURING PCR, INSERTED INTO A PLASMID IF NECESSARY , AND CLONED. RESTRICTION SITED CAN BE ADDED TO A FRAGMENT OF DNA, INSERTED INTO A PLASMID, AND CLONED. SCIENTISTS CAN TEST FOR THE PRESENCE OF A SPECIFIC SEQUENCE OF DNA IN A SAMPLE (USE AS A TEST TO SEE IF THE DNA WAS TRULY INCORPORATED INTO A NEW ORGANISM). SCIENTISTS CAN AMPLIFY A PIECE OF RECOMBINANT DNA TO USE IN SEQUENCING REACTIONS . What are some other uses of PCR? Sequencing of DNA Paternity testing Forensic proof of presence at a crime scene Identifying the sequence of specific genes Observing the evolution of a disease-causing agent (or other organism) Identifying unknown samples Testing for the presence of bacteria or a virus (as a sign of infection in a patient) Testing for the presence of cancer cells Testing for the presence of genetic defects