MOLECULAR TECHNIQUES

EJC H2 Biology 1 T1 W6 Molecular Biology Techniques Genetics and Inheritance – Molecular Techniques 1. Introduction Molecular biology techniques are common methods used in molecular biology, biochemistry, genetics and biophysics which generally involve manipulation and analysis of DNA, RNA, proteins, and lipids. Since around 1960, molecular biologists have developed methods to identify, isolate, and manipulate mo lecular components in cells including DNA, RNA, and proteins. One important tool used in molecular biology techniques especially in molecular cloning is the restriction enzyme . These enzymes are found in bacteria and provide a defense mechanism against invading viruses. It is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites. Several techniques used in the field of molecular biology include:  Polymerase chain reaction (PCR)  Gel electrophoresis  Macromolecule blotting and probing e.g. Southern blotting , Northern blotting, Western blotting and Eastern blotting  Expression cloning 2. Learning Outcomes (k) describe the principles and procedures of these molecular techniques: i. polymerase chain reaction (including its advantages and limitations) ii. gel electrophoresis iii. Southern blotting and nucleic acid hybridisation Use the knowledge gained in this section in new situations or to solve related problems. 3. References i. T.A.Brown (2010) Gene cloning & DNA Analysis, 6 th Edition, Wiley -Blackwell. Chapter 1 to 7, 9. ii. Watson, Mayers, Caudy, Witkowski (2007), Recombinant DNA, Genes and Genomes – A short Course. Cold Spring, Harbor Lab Press. Chapter 4 iii. Campbell and Reece (2011) Biology, 9th Edition, Pearson Education. Chapter 20. iv. Turner, McLennan, Bates and White (2001) Molecular biology, 2 nd Edition, BIOS. Sections G, H, I and J. v. Green, Stout and Taylor (1997), Biological Science Volumes 1 and 2, 3rd Edition. vi. Hoh Yin Kiong (2003), Longman A-Level Course in Biology, Core Syllabus Volume 1 (for AS and A-Level) vii. Weaver (2008), Molecular Biology(4th ed.). McGraw-Hill. viii. http://www.dnalc.org/resources/spotlight/index.html (Cold Spring Harbor Laboratory DNA Learning Centre. Look for Polymerase Chain Reaction).

EJC H2 Biology 2 T1 W6 Molecular Biology Techniques Contents 1. Introduction ............................................................................................................... 1 2. Learning Outcomes.................................................................................................... 1 3. References ................................................................................................................. 1 4. Molecular Biology Techniques .................................................................................. 3 A. Polymerase Chain Reaction………………………………………………….…………………………...3 B. Gel Electrophoresis………………………………………………………………………………..12 C. Southern Blotting………………………………………………………………………….…….…15 5. Restriction Enzymes ................................................................................................ 17 6. Restriction Fragment Length Polymorphism ........................................................... 19 7. Appendix .................................................................................................................. 27

EJC H2 Biology 3 T1 W6 Molecular Biology Techniques 4. Molecular Biology Techniques A. Polymerase Chain Reaction What is PCR? Polymerase Chain Reaction (PCR) is a technique that allows amplification (making many copies) of a specified segment of DNA in vitro (in a test tube).  Amplifying DNA through PCR has extensive applications such as in basic research, human genetics testing and forensics. Until the mid-1980s, the only way to make many copies of DNA was to insert the DNA pieces into bacteria and select the desired one from many different colonies growing on a plate (this takes several weeks!). In 1985, Kary Mullis (shown right) invented a precise and radical new method of selecting and amplifying a section of DNA – the PCR which takes less than a few hours to complete. The revelation came to this eccentric character and avid surfer on a drive in northern California with his girlfriend. Kary Mullis was a recipient of the 1993 Nobel Prize in Chemistry. His award has drawn controversy in that he was not the first to describe nor ‘invent’ the processes used in PCR but others have defended that it was Mullis who first put the processes together into a system for molecular biology. Prior Knowledge Required 1. Structure of DNA:  DNA strands unzip and separate at high temperature as hydrogen bonds between the complementary base pairs are broken (denature).  DNA strands anneal through formation of hydrogen bonds between complementary base-pairs upon cooling. 2. DNA replication:  Primers are needed during DNA replication in vitro as DNA polymerase needs an existing free 3’ hydroxyl group for chain extension to take place. Notes to self

EJC H2 Biology 4 T1 W6 Molecular Biology Techniques PCR reaction mixture contains the following reagents/components: PCR component Details Template DNA  DNA containing the segment or sequence of bases to be amplified. This can usually be genomic DNA, plasmid, or fragmented DNA. Oligonucleotide primers (oligo = short)  Synthetic single-stranded DNA typically 20-30 nucleotides long.  Needed to initiate DNA synthesis.  The primers ( forward and reverse primers) are complementary to sequences at the 3’ ends of the target DNA sequence to be amplified.  Primers serve to:  mark out the section of DNA to be amplified by attaching to complementary bases on the 3’ ends of the target DNA sequence;  provide a free 3’-OH end for DNA polymerase to add DNA nucleotides onto to elongate a new strand of DNA.   Knowledge of the sequences flanking / at the ends of the DNA segment of interest is thus crucial to artificially synthesise the primers.  Primers are present in large excess in reaction mixture to increase likelihood of primers binding to target DNA (decrease likelihood of template DNA strands reannealing to each other again).  The primers become part of the amplified segments. Taq polymerase  A thermostable (resistant to denaturation at high temperatures) DNA polymerase isolated from a thermophilic prokaryote living in hot springs, Thermus aquaticus (Taq).  Enzyme is stable at 95C and works optimally at 72C.  It binds to the primer at the 3’ end.  Using the DNA template, Taq polymerase catalyses the formation of a phosphodiester bond between the adjacent nucleotides, synthesising new DNA strands via complementary base -pairing in the 5' to 3' direction. Deoxyribonucleo side triphosphates (dNTPs)  dATP, dTTP, dCTP and dGTP are substrates for DNA replication. Template DNA strands Primers Forward primer Reverse primer

EJC H2 Biology 5 T1 W6 Molecular Biology Techniques Buffer containing Mg2+  Mg2+ is a cofactor1 for proper polymerase function. PCR Procedure Protocol: 1. Place all the components into a PCR tube, and place the tube into the thermocycler (Fig. 1). Run a standard programme that involves heating the tube to different temperatures for different periods of time. Entire process is now fully automated. 2. Steps during PCR: The following three steps constitute 1 cycle. The cycle is then repeated (Figs. 2, 3). Step Details 1. Denaturation  A brief heat treatment (up to 95C) to denature/unzip and separate the two strands of DNA double helix. This exposes the bases for complementary base pairing required in steps 2 and 3. 2. Primer annealing  Cooling of the DNA (e.g. 65C) in presence of a large excess of DNA primers allows their specific annealing to complementary sequences at the 3’ ends of each of