Lecture 2 - Gene Analysis.pdf

Lecture 2 - Gene Analysis Dr Federico Rojas [email protected] Agarose gel electrophoresis Nucleic acids are negatively charged (because of their phosphate backbone), so they migrate toward the positive electrode. The gel acts like a sieve, selectively retarding the movement of larger mo...

Lecture 2 - Gene Analysis Dr Federico Rojas [email protected] Agarose gel electrophoresis Nucleic acids are negatively charged (because of their phosphate backbone), so they migrate toward the positive electrode. The gel acts like a sieve, selectively retarding the movement of larger molecules. Visualizing DNA bands ultraviolet (UV) irradiation. Ethidium bromide fluoresces under UV light Visualizing DNA bands in an agarose gel by EtBr staining and ultraviolet (UV) irradiation Agarose percentage The composition of the gel determines the sizes of the DNA molecules that can be separated. A 0.5 cm-thick slab of 0.5% agarose, which has relatively large pores, would be used for molecules in the size range 1 to 30 kb. Agarose % (w/v) Resolution 0.50 % 1,000 – 30,000 bp 0.70 % 800 – 12,000 bp 1.00 % 500 – 10,000 bp 1.20 % 400 – 7,000 bp 1.50 % 200 – 3,000 bp 2.00 % 50 – 2,000 bp Techniques to study gene/DNA structure (sequence) 1. Restriction Mapping/Restriction 2. Fragment Length Polymorphism (RFLP) 3. Polymerase Chain Reaction 4. Southern Analysis 5. DNA Sequencing Restriction mapping Mapping the positions of different restriction sites in a DNA molecule To construct a restriction map, a series of restriction digests must be performed. 1. The number and sizes of the fragments produced by each restriction endonuclease must be determined by gel electrophoresis. 2. Information must then be supplemented by a series of double digestions (two restriction endonucleases at once) Marker Comparing the results of single and double digests will allow many, if not all, of the restriction sites to be mapped 33- 2415- 9- The only possibility is: XbaI XhoI XhoI+XbaI Restriction mapping - Estimation of the sizes of DNA molecules Gel electrophoresis separates different-sized DNA molecules, with the smallest molecules travelling the greatest distance toward the positive electrode. Restriction fragment length polymorphisms (RFLP) analysis An RFLP is a sequence variation that changes a restriction site Human breast cancer susceptibility gene BRCA1: a significant number of the women who suffered from the disease all possessed the same version of an RFLP called D17S74. Polymerase chain reaction (PCR) Kary Mullis invented the polymerase chain reaction (PCR) Thermostable DNA polymerases In the 1960s, Thomas Brock was a biologist at Indiana University. He started a field research station in Yellowstone National Park. At the time, scientists believed that bacteria optimally lived at about 55°C, and that nothing lived above 73°C The bacteria: Thermus aquaticus, which contains the DNA polymerase that ultimately became the backbone of PCR: Taq Polymerase Polymerase chain reaction (PCR) - 1 PCR uses repeated rounds of strand separation, hybridization, and synthesis to amplify DNA 1. Denaturation DNA at 94°C 2. Annealing at 50–60°C 3. Synthesis of new DNA at 74°C Molecular Biology of the Cell, 6th edition Polymerase chain reaction (PCR) - 2 Repeat the cycle 25–30 times Molecular Biology of the Cell, 6th edition Polymerase chain reaction (PCR) - 3 Because a heat-resistant polymerase is used, the reaction can be repeated continuously without the addition of more enzyme. Each cycle doubles the copy number of the amplified gene: 10 cycles ideally produce: 1,024 (210) copies 30 cycles yield (210x3) = 109-fold amplification PCR uses Amplification of the desired sequence prior to cloning Diagnostic and Forensic applications Species identification (meat scandals) Identification of disease alleles Gene expression (next lecture) Sensitivity – theoretically only one target molecule required Specificity – The polymerase chain reaction allows to obtain a pure sample of a gene. Southern blotting Allows specific fragments to be identified in a complex mixture Named after Ed Southern. As well as colony hybridisation analysis, there are also occasions when it is necessary to use hybridisation probing to identify which of a series of restriction fragments contains a gene of interest B = BamHI restriction site. Southern blotting - 2 The DNA to be analysed is digested with a restriction endonuclease, and the digested DNA fragments are separated by gel electrophoresis. Southern blotting (continue) The same method can also be used for the transfer of: RNA molecules (Northern transfer) or Proteins (Western transfer) So far, no one has devised Eastern transfers. Nucleic acids are generally detected by hybridisation with homologous sequences. renature to form double-stranded molecules DNA sequencing Determination of the order of bases in a strand of DNA (in a 5’ to 3’ direction) 1. Sanger dideoxynucleotide method of DNA sequencing Based on the principle of premature termination of DNA synthesis from the inclusion of chain-terminating dideoxynucleotides. Requires: Single-stranded DNA template Primer dNTPs (dNTPs – dATP, dCTP, dGTP, and dTTP) ddNTPs dideoxynucleotides (low concentration) DNA polymerase Frederick Sanger Sanger sequencing Each of the four dideoxynucleotides is labelled with a different fluorescent dye, so their incorporation into DNA can be monitored. The incorporation of a dideoxynucleotide stops further DNA synthesis because no 3′ hydroxyl group is available for the addition of the next nucleotide. X Sanger sequencing - 2 Thus a series of labelled DNA molecules is generated, each terminating at the base represented by a specific fluorescent dideoxynucleotide. Possible to obtain over 750 bp of sequence per experiment. Genes are longer than this: carry out two or more chain-termination experiments, directed at different parts of a gene, in order to build up the complete sequence. The chain-termination (Sanger) method was also used to obtain the first complete genome sequences. Challenging 3200 Mbases Furthermore, no sequencing method is entirely accurate it is necessary to sequence each region of a genome multiple times, in order to identify errors present in individual sequence reads With the chain-termination method, at least a fivefold sequence depth or coverage is required, which means that every nucleotide is present in five different reads. Cycle Sequencing The key difference with traditional Sanger method: the employment of a thermo stable DNA polymerase. The advantage: the sequencing reaction can be repeated over and over again in the same tube by heating the mixture to denature the DNA and then allowing it to cool down to anneal the primers and polymerise new strands. Therefore less template DNA is needed than for conventional sequencing reactions.