tailieunhanh - Molecular Biotechnology-Lession 3: Basic techniques in DNA technology

Polymerase Chain Reaction is widely held as one of the most important inventions of the 20th century in molecular biology. Small amounts of the genetic material can now be amplified to be able to a identify, manipulate DNA, detect infectious organisms, including the viruses that cause AIDS, hepatitis, tuberculosis, detect genetic variations, including mutations, in human genes and numerous other amplifying a single or some piece of DNA generating thousands of million copies of DNA Total copies = 2n where n is the number of cycles to copy DNA So basically it is the cycles of heating and cooling (thermal cycli. | Molecular Biotechnology Tran Ngoc Duc, PhD VIETNAM NATIONAL UNIVERSITY AT HO CHI MINH CITY INTERNATIONAL UNIVERSITY SCHOOL OF BIOTECHNOLOGY Basic techniques in DNA technology Polymerase chain reaction (PCR) Gel electrophoresis Primer Design RT-PCR Digestion Southern blot/Northern blot Detectable signal based techniques Site-directed mutagenesis Polymerase chain reaction PCR and Primer Design Developed in 1983 by Kary Mullis Polymerase Chain Reaction is widely held as one of the most important inventions of the 20th century in molecular biology. Small amounts of the genetic material can now be amplified to be able to a identify, manipulate DNA, detect infectious organisms, including the viruses that cause AIDS, hepatitis, tuberculosis, detect genetic variations, including mutations, in human genes and numerous other tasks. Components in PCR reaction Template DNA Primers dNTPs Taq DNA polymerase Buffer Step 1: Denature DNA, 95oC, 5min Step 2: Annealing: - Denature DNA, 950C, | Molecular Biotechnology Tran Ngoc Duc, PhD VIETNAM NATIONAL UNIVERSITY AT HO CHI MINH CITY INTERNATIONAL UNIVERSITY SCHOOL OF BIOTECHNOLOGY Basic techniques in DNA technology Polymerase chain reaction (PCR) Gel electrophoresis Primer Design RT-PCR Digestion Southern blot/Northern blot Detectable signal based techniques Site-directed mutagenesis Polymerase chain reaction PCR and Primer Design Developed in 1983 by Kary Mullis Polymerase Chain Reaction is widely held as one of the most important inventions of the 20th century in molecular biology. Small amounts of the genetic material can now be amplified to be able to a identify, manipulate DNA, detect infectious organisms, including the viruses that cause AIDS, hepatitis, tuberculosis, detect genetic variations, including mutations, in human genes and numerous other tasks. Components in PCR reaction Template DNA Primers dNTPs Taq DNA polymerase Buffer Step 1: Denature DNA, 95oC, 5min Step 2: Annealing: - Denature DNA, 950C, 30s-1min - Annealing, 55-60oC, 1min Step 3: Extension, 70-72oC, 30s-1min Go to step 2, 32 cycles Step 4: Holding, 4oC, 0s Steps programmed in PCR Exponentially amplifying a single or some piece of DNA generating thousands of million copies of DNA Total copies = 2n where n is the number of cycles to copy DNA So basically it is the cycles of heating and cooling (thermal cycling) Applications of PCR DNA cloning DNA based phylogeny Functional analysis of gene Hereditary diseases Forensic sciences Infectious diseases 2. Gel electrophoresis A method for separating mixture of charged molecules such as DNA, RNA or proteins under an electric field when an electric field is applied to them The gel acts like a sieve for separation of molecules according to their sizes and electric charges Gel is made of agarose for separating large molecules of DNA, and of acrylamide for protein separation or small DNA or RNA Proteins, unlike nucleic acids, can have varying charges and complex shapes, .