15) Molecular Techniques 9744 2019

Prepared by: Mrs Selvamani Nair & Ms Eva Hor Raffles Institution (2019) 1 Genetics and Inheritance (9744) Molecular Techniques 2019 Describe the principles and procedures of the polymerase chain reaction (PCR) (including its advantages and limitations) Describe the principles and procedures of gel electrophoresis . Polymerase Chain Reaction  Amplifies DNA from a limited source of DNA so that there is sufficient amount for analysis. * Process: (3 steps) 1. 1. Denaturation: Double stranded DNA denatures into single stranded DNA by heating to 95C 2. as weak hydrogen bonds between complementary bases of each strand is 3. broken due to increased molecular vibrations 2. Primer annealing: Each primer anneals specifically to the 3’end of each single stranded target DNA sequence via complementary base pairing when the temperature is lowered to 64C 3. Extension: Taq polymerase synthesizes the complementary DNA strand from the free 3OH’ end of the DNA primer by catalyzing the formation of phosphodiester bonds between dNTPS when the temperature is increased to 72C * Advantages: 1. Only a minute amount of DNA is required to carry out PCR as with each round of PCR, the number of copies of target DNA is doubled. Thus the number of desired sequence increases exponentially and there will be sufficient DNA for analysis. 2. Use of thermostable (i.e. resistant to denaturation at high temperatures) Taq polymerase allows PCR to be automated so DNA can be amplified very quickly. * Limitations: 1. Taq polymerase lacks 3’ to 5’ proofreading ability. Hence errors occurring early in the PCR reaction will get compounded with each subsequent replication cycle. 2. Knowledge of sequences flanking (i.e. at the 3’ ends of) the target sequence is required in order to design appropriate primers. 3. Taq polymerase tends to ‘fall off’ the DNA template before chain extension is complete if the strand is too long. Hence there is a limit to the size of DNA fragment (~3kb) to be amplified. 4. Minute amounts of contaminant DNA can be exponentially amplified along with target DNA and affect the reliability of the results. Agarose gel electrophoresis  separates DNA based on fragment size * Steps: 1. A slab of agarose gel is placed in a buffer solution contains ions which allows the conduction of electricity when the current is turned on. 2. The DNA sample is mixed with a dense loading dye containing glycerol & 2 coloured dyes. Glycerol makes the DNA sample denser than the buffer solution so that the DNA sample can sink to the bottom of the well. 3. Since DNA is invisible, the dyes colour the DNA sample and will indicate if the DNA has been loaded correctly into the well. (NB: Loading dyes do not bind to the DNA.) 4. One dye (corresponds to a 100bp DNA fragment) and often runs ahead of the DNA sample and gives an indication of when electrophoresis must be stopped so that the samples do not run out of the gel. The other dye (corresponds to a 1100bp DNA fragment) and gives an indication of the position of the larger fragments on the gel. 5. The 2 coloured dyes thus act as visual markers which help to monitor the progress of the migration of the invisible DNA fragments in the gel during electrophoresis. 6. DNA samples are pipetted into the wells in the gel near the negative electrode. 7. A DNA ladder (i.e. DNA molecular weight markers) which contains DNA fragments of known sizes, is run in one of the lanes and acts as a standard for which to compare fragments of unknown size in the sample. 8. Negatively charged DNA is attracted towards the positive electrode (anode) when subjected to an electric current. 9. The agarose gel matrix made of a meshwork of polymer fibres which impedes movement of longer fragments more than shorter fragments. The longer fragments thus migrate more slowly compared to shorter fragments, leading to a banding pattern observed on the gel. 10. Before the loading dye reaches the end of the gel, the current is turned off. 11. To visualize the bands, the gel can be treated with a staining dye that binds DNA (e.g. ethidium bromide, a carcinogen) and fluoresces under uv light. 12. Thus a) the fragment size can be estimated (based on position of the band relative to bands in the molecular weight marker) and b) the amount of DNA can possibly be estimated (based on intensity & thickness of the band). PCR Components * Template DNA DNA containing the target sequence to be amplified * Primers synthetic single-stranded DNA fragment (20-30 nucleotides long) needed to initiate DNA synthesis by providing a free 3’OH group for Taq polymerase to bind to and extend  2 different primers are required. Each is complementary to the sequence at 3’end of each single stranded target DNA sequence.  are required in large excess to increase the likelihood of them binding to target DNA sequences (relative to template strands binding each other) * Taq polymerase  thermostable DNA polymerase which is resistant to denaturation at high temperature * Deoxyribonucleotides (dNTPs)  substrates for DNA replication made up of dATP, dTTP, dCTP and dGTP * Buffer  contains cofactor, Mg2+, for proper polymerase function * Number of copies of double stranded DNA = 2n (where n is the number of cycles) * Number of (single) strands of DNA = 2n+1 (where n is the number of cycles) End of n cycle where n is 1 2 3 4 Copies of ds DNA 2 4 8 16 Strands of ss DNA 4 8 16 32 The top line corresponds to fragments that are 1100bp and the bottom line corresponds to 100bp fragments. DNA molecular weight marker acts as standard to compare to fragments of unknown sizes Note: Bands are only visible after treating with staining dye such as ethidium bromide (visible under UV light). NB: The higher the concentration of agarose, the finer the pores in the meshwork. Thus smaller fragments can be effectively separated. DNA strand End of: 1st cycle 2nd cycle 3rd cycle 4th cycle TAC ATT

Prepared by: Mrs Selvamani Nair & Ms Eva Hor Raffles Institution (2019) 2 Describe the principles and procedures of Southern blotting and nucleic acid hybridisation Southern blotting: a nucleic acid hybridisation technique used in RFLP analysis Tool to detect specific nucleotide sequences within a sample of DNA Steps: 1. (Continued from Gel electrophoresis) 1. Gel slab is placed on top of the sponge and under a nitrocellulose membrane. A stack of paper towels placed on top of nitrocellulose membrane. These are placed in a tray of alkaline solution. A heavy weight is placed above the paper towels. 2. Absorbent paper towels draw the solution towards themselves and the alkaline solution den