Molecular Biology Techniques PDF

Molecular Biology Techniques Lecture 12 Molecular Biology Department of Biology Faculty of Mathematics and Natural Sciences Universitas Indonesia Contents...

Molecular Biology Techniques Lecture 12 Molecular Biology Department of Biology Faculty of Mathematics and Natural Sciences Universitas Indonesia Contents Nucleic PCR acid Labelling DNA Sequencing Blotting (Western, and Southern) DNA Sequencing Determining sequence of bases in DNA that enables the processive identification of each base in turn. Three main requirements to achieve processive identification: DNA fragmens need to be prepared in a form suitable for sequencing The technique used must achieve the aim of presenting each base in turn in a form suitable for identification The detection method must permit rapid and accurate identification of the bases. DNA Sequencing: principles Sequencing technique will Detection step is the final stage generate nested fragments, which of sequencing procedure, is overlapping fragments that usually involves separation of terminate at different bases and fragments on a polyacrylamide differ in length by one nucleotide. gel. Two main methods for DNA sequencing: enzymatic and chemical method.  Preparation of DNA fragments Two strategies for sequencing large stretches of DNA Ordered Sequencing Strategy Shotgun Sequencing Method Fragments are tracked and their relative order is noted as the project progress, and then put together by reference to the order of the fragments. Fragments are generated and processed at random, assembly of the sequence is then carried out by searching for sequence overlaps using a computer. DNA Sequencing Maxam-Gilbert Sanger-Coulson (chemical) (enzymatic) sequencing sequencing Maxam-Gilbert (chemical) sequencing Starting material: defined fragment of DNA. Applicable to any DNA fragment (doesn’t need to be cloned in a plasmid vector). Theory: given the large number of molecules and different reactions, this process will produce a set of nested fragments. a. DNA is radiolabelled with 32P at 5’ ends of each strand and then the strands are denatured, separated, and purified to give a population of labelled strands for the sequencing reactions. b. Chemical modification of bases in DNA strand in series of four to five reactions with different specificities, with on average, one base being affected per molecule. c. Modified bases are removed from their phosphodiester backbone and the strands cleaved at these positions using piperidine. d. The process produces a set of fragments differing in length by one nucleotide, labelled at their 5’ termini. Maxam- Gilbert (chemical) sequencing *ATTGACTTAGCC*ATTGACTTAGCC *ATTGACTTA *ATTGACTTA *ATTGACTTAGCC*ATTGACTTAGCC *ATTGACTTAGC *ATTGACTTAGC *ATT *ATTGACTT *ATTGACTTAG *ATTGACTTAG *ATTG *ATTGACT *ATTGA *ATT *ATTGAC *ATTGA *AT *A Sanger-Coulson (enzymatic) sequencing Similar END RESULT, totally different PROCEDURE. More complicated and usually involves subcloning into different vectors like plasmid. Another method is to clone the DNA into vector such as bacteriophage M13 which produces ssDNA during infection→provides suitable substrate for the sequencing reactions. Sanger-Coulson (enzymatic) sequencing a. A Primer is annealed to a ssDNA template. b. The Klenow fragment of DNA polymerase I is used to synthesize a copy of the DNA. A radiolabelled dNTP (usu. [α-35S]dNTP, solid circles) is incorporated into the DNA. c. Chain termination occurs when dideoxynucleoside triphosphate (ddNTP) is incorporated. d. A series of four reactions, each containing one ddNTP in addition to the four dNTPs required for chain elongation, generates a set of radiolabelled fragments. DNA synthesis requires a 3´-OH to make the next phosphodiester bond during DNA synthesis normal dNTP  H ddNTPs block formation of the next phosphodiester bond during DNA synthesis. A 3´-OH on the last ribose is needed for DNA synthesis. A mixture of dNTPs and ddNTPs are used in DNA sequencing Sanger-Coulson (enzymatic) sequencing Reading DNA Sequence a. An autoradiograph of part of a sequencing gel. b. A tracing of the autoradiograph. Each lane corresponds to a reaction containing one of the four ddNTPs used in the chain- termination technique for DNA sequencing. Read from the bottom of gel. Electrophoresis and reading of sequences Separation → achieved by PAGE. Gels are 0.5 mm thick, usually contain 6-20% polyacrylamide and 7 M urea, which acts as a denaturant to reduce the effects of DNA 2’ structure → important, because fragments that differ in length by only 1 base are separated. 1. Gel is run at hi-power settings → heat up 60-7-oC → maintain denaturing condition. 2. After running gel, it is removed and may be dried onto a paper sheet and then exposed to X-ray film. 3. Emissions from the radioactive label sensitize the silver grains, which turn black when the film is developed and fixed → autoradiograph (see prev. slide). Polyacrylamide gel electrophoresis is used to visualize the results of the sequencing reaction Automation of DNA sequencing Enables large-scale production. Improvements in sample preparation and handling, with robotic processing enabling hi-volume throughput. Linear continuous capillary electrophoresis techniques. Without the ability to analyse sequence data, the sequence remains essentially silent/uninformative → dealt with BIOINFORMATICS! Automated DNA sequencing with fluorescent dyes coupled to each reaction Fluorescent dye coupled to reaction allows visualization of di-deoxy termination events by means of a laser that detects the colored product. This shows four different reactions as done with the old manual sequencing. Automated DNA sequencing output 4 reactions carried out in one tube The genomes of many organisms have been sequenced Contents Nucleic PCR acid Labelling DNA Sequencing Blotting (Western, and Southern) PCR : A Molecular Xerox Machine for DNA Some techniques to analyze DNA and RNA are limited by the small amounts of test nucleic acid available. Polymerase chain Can replicate a reaction (PCR) target DNA from a rapidly increases few copies to the amount of DNA millions in a few in a sample. hours. What is PCR? ▪ It is a molecular technology aim to amplify a single or few copies of the DNA to thousands or millions of copies. ▪ Developed in 1983 by Kary Mullis, PCR is now a common and important technique used in biological research labs for a variety of applications. ▪ In 1993, Mullis was awarded the Nobel prize in Chemistry along with Michael Smith for his work on PCR. NEW AUTOMATED PCR OLD PCR Waterbath with 3 different temperatures Three Basic Steps that Cycle Denaturation Priming Extension Heat to 98oC to Primers added in a 72oC separate into two concentration that DNA polymerase strands. favors binding to extend the Cool to between the complementary molecule. 50oC and 65oC. strand of test DNA. Prepares the two strands for synthesis. The amplified DNA can then be analyzed. PCR PCR increases the yield of DNA exponentially PCR requirement Component Function DNA template Any source of DNA that provides one or more target molecules can in principle be used as a template for PCR. Purity and quantity is important. Primers Specific to the section of DNA to be copied. Forward and reverse. Good primers are important. Polymerase Enzyme that reads original DNA sequence and makes a complementary copy. DNA pol. from Thermus aquaticus is stable at 95oC (Taq) Pyrococcus furiosus (Pfu pol.), Thermus thermophilus (Tth pol.), Thermus flavus (Tfl pol.) dNTPs Nucleotides used to create the complementary strand of DNA. PCR buffer, MgCl2, Enables the reaction to take place. Water  Designing PCR primers 1. Primers should be 17-28 bases in length; 2. Base composition should be 50-60% (G+C); 3. Primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming; 4. 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesized preferentially to any other product; 5. Primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided; 6. Tms between 55-80oC are preferred. Designing PCR primers 1. Primers should be 17-28 bases in length; Increase uniqueness. 2. Base composition should be 50-60% (G+C); Also affecting: - Melting temperature - Stability 3. primers should end (3') in a G or C, or CG or GC (GC clamp): this prevents "breathing" of ends and increases efficiency of priming; The presence of G or C bases within the last five bases from the 3' end of primers (GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C bases.  Designing PCR primers 4. 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesized preferentially to any other product; 5. Primer self-complementarity (ability to form 2 o structures such as hairpins) should be avoided; Self-Dimer Hairpin loop ΔG Positive: not stable ΔG Negative: stable  Designing PCR primers 6. Tms between 55-80oC are preferred; The temperature at which one half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability Primers with Tm 52 – 58 oC generally produce the best results. The GC content of the sequence gives a fair indication of the primer Tm. The difference between Tm forward & Tm reverse should not bigger than 3oC. Basic Tm calculation Tm= (wA+xT)*2 + (yG+zC)*4 (Marmur J and Doty P (1962) J Mol Biol 5:109-118) Catatan:w,x,y,z adalah jumlah basa  Annealing temperature (Ta) Ta Opt = 0.3 x(Tm of primer) + 0.7 x(Tm of product) - 14.9 Tm product can be calculated if the sequence of product is known where Tm of primer is the melting temperature of the less stable primer-template pair Tm of product is the melting temperature of the PCR product Rychlik W., Spencer W.J., Rhoads R.E., Spencer W.J., Rhoads R.E., Rhoads R.E. Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res. 1990;18:6409–6412. Generally : Tanneal = Tm_primer – 5C Hot start PCR A modified form of PCR which avoids a non-specific amplification of DNA by inactivating the taq pol. at lower temperature. The enzyme is not active in low temp. by adding specific antibodies that block Taq-polymerase at annealing temperature. But when the temperature raises for amplification to 72oC, the specific antibody detaches from Taq-polymerase and the amplification with greater specificity starts. Hot Start PCR significantly reduces nonspecific priming.  Real Time (reverse transcriptase) PCR RT-RT-PCR or just RT-PCR. Tissue / cells To quantitate differences in mRNA extract RNA expression → to have an idea about gene expression (need to be compared to the expression of copy into cDNA reference genes, eg. housekeeping (reverse transciptase) genes). do real-time PCR Limited amount of mRNA Small amount of tissue/cells analyze results Precious reagents. Real Time (reverse transriptase) PCR To quantitate differences in mRNA expression ? PCR / reverse transcriptase PCR and subsequent analysis by agarose gels → more qualitative Northern → agarose, blot → qualitative In situ hybridization → qualitative Real Time PCR gives quantitative results Example (1) : the expression of genes encoding AIR12, Putative early nodulin-like 2, Rhicadhesin receptor & Blue copper domain proteins in 2 different samples (2HA & SKL plants) Example (2) : the expression of genes encoding Serine carboxypeptidase in 4 organs (leaf, cotyledon, root & root tip) in inoculated & uninoculated plants. Serine carboxypeptidase 16 14 Relative expression 12 10 8 6 4 2 0 Leaf Ctrl Leaf Inoc Cotyl Ctrl Cotyl Inoc Root Root Root Tip Ctrl Root Tip Segment Segment Inoc Ctrl Inoc  Real Time PCR vs PCR Web tools to design RT-PCR primers https://www.genscript.com/ssl- bin/app/primer http://sg.idtdna.com/scitools/Applicati ons/RealTimePCR/ http://www.quantprime.de/ Methods: Using fluorescent intercalating agent (eg. SYBR green fluorescence) Using Taqman probe  RT-PCR using SYBR Green SYBR green is a fluorogenic minor groove binding dye that exhibits little fluorescence when in solution but emits a fluorescent signal upon binding to double-stranded DNA (fluorescent intercalating agent).  RT-PCR using Taqman probe The light emitted from the dye in the excited state is received by a Before the probe is met with the Taq polymerase, computer and shown on a graph energy is transferred from a short-wavelength display, such as this, showing fluorophore (green) to a long-wavelength fluorophore PCR cycles on the X-axis and a (red). When the polymerase adds nucleotides to the logarithmic indication of intensity template strand, it releases the short-wavelength on the Y-axis. fluorophore, making it detectable and the long- wavelength undetectable. Taqman probes The PCR is prepared as usual, and the reporter probe is added. As the reaction commences, during the annealing stage of the PCR both probe and primers anneal to the DNA target. Polymerization of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its 5'-3'- exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence. Fluorescence is detected and measured in a real-time PCR machine, and its geometric increase corresponding to exponential increase of the product is used to determine the quantification cycle in each reaction. Nested PCR PCR site directed mutagenesis Common PCR Restriction fragment length polymorphisms (RFLPs) Southern Blot Consider two alleles of a gene. Allele A has 3 BamHI sites, while allele a has only two BamHI sites. probe HpaI Digest Nor- Variants mal 1 2 3 70% of carriers of the sickle cell gene have a 13.0 kb HpaI fragment. 30% of carriers have 7.0 kb HpaI fragment.  PCR applications Marker development: RAPD PCR, RFLP PCR, AFLP PCR, etc. DNA fingerprinting using molecular marker. Site directed mutagenesis → using primer that contains the mutations. Medical applications: For prenatal diagnosis, PCR used to amplify DNA from fetal cells obtained from amniotic fluid. Tay-Sachs disease, phenylketonurea, cystic fibrosis, hemophilia, Huntingdon's disease, Duchenne muscular dystrophy (DMD). the direct detection of HIV genomes in patient blood before the appearance of HIV antibodies Forensic application  Variable Number of Tandem Repeat (VNTR) analysis is commonly used in forensics VNTR is based on hypervariable microsatellite sequence polymorphisms within the human genome. These sequences (e.g., CACACA …) are found in many locations in the human genome and vary greatly from person to person. Using VNTR to compare forensic and suspect samples Individuals A & C are excluded by this analysis. The samples from individual B will be subjected to further tests. PCR Results Detection Contents Nucleic PCR acid Labelling DNA Sequencing Blotting (Western, and Southern)  Labelling the Nucleic Acid 1)Keeping track (tracing) of small amounts of nucleic acid → low specific activity is suffice 2) To produce highly radioactive nuc. acid molecules for hybridization (radioactive probes) → high specific activity is necessary Types of label: Radioactive (radiolabeling) tritium (3H), carbon-14 (14C) → low energy emitter sulphur-35 (35S) → medium energy emitter phosphorus-32 (32P) → high energy emitter → more hazardous Non radioactive Fluorescent dyes Enzyme-linked labels → Elisa Radioactive molecules → dNTP labelled with 3H or 32P Labelling the Nucleic Acid – End Labelling If the ATP donor is radioactively labelled, this produces a labelled nucleic acid of relatively low specific activity, as only the termini of each molecule become radioactive.  Labelling the Nucleic Acid – Nick translation If labelling DNA by nick translation. (a) A single-strand nick is introduced into the phosphodiester backbone of a DNA fragment using DNase I (low conc). (b) DNA polymerase I then synthesizes a copy of the template strand, degrading the non- template strand with its 5→3 exonuclease activity. If [α-32P]dNTP is supplied this will be incorporated into the newly synthesized strand (solid circles).  Labelling the Nucleic Acid – Primer extension Labelling DNA by primer extension (oligolabelling). (a) DNA is denatured to give single-stranded molecules. (b) An oligonucleotide primer is then added to give a short double-stranded region with a free 3-OH group. (c) The Klenow fragment of DNA polymerase I can then synthesise a copy of the template strand from the primer, incorporating [α-32P]dNTP (solid circles) to produce a labelled molecule with a very high specific activity. Nucleic Acid Hybridization Contents Nucleic PCR acid Labelling DNA Sequencing Blotting (Western, and Southern) Blotting Southern Blot  Southern Blot - Method 1. Restriction 2. Gel electrophoresis 3. DNA denaturation (alkaline method, NaOH) – denature dsDNA 4. Blotting to nitrocellulose membrane 5. Fixation (using vacuum or oven 80oC or UV radiation) to permanently attach DNA to membrane 6. Hybridization 7. Wash excess probe Western Blot Western Blot – Detection, Why not radiolabeling? Membrane-bound proteins are generally detected using secondary antibodies that are labeled with radioisotopes or colloidal gold, or that are conjugated to fluorescent molecules (fluorophores) or an enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP). Early blotting systems used 125I-labeled reagents similar to those used in radioimmunoassays. These systems provide sensitive results, but the special handling and disposal problems of 125I reagents have discouraged continued use of this technique. Western Blot Mechanism of detection chemistries. In each method of western blot detection, a detectable signal is generated following binding of an antibody specific for the protein of interest. In colorimetric detection (A), the signal is a colored precipitate. In chemiluminescence (B), the reaction itself emits light. In fluorescence detection (C), the antibody is labeled with a fluorophore.  Gel Electrophoresis Nondenaturing GE / Native Agarose GE Denaturing gradient GE Polyacrylamide GE Pulse field GE Denaturing gradient GE / DGGE using chemical gradient DNA is subjected to increasingly extreme denaturing conditions, the melted strands fragment completely into single strands. Able to recognise the differences in DNA sequences or mutations of various genes. sequence differences in fragments of the same length often cause them to partially melt at different positions in the gradient and therefore "stop" at different positions in the gel. By comparing the melting behavior of the polymorphic DNA fragments side-by side on denaturing gradient gels, it is possible to detect fragments that have mutations in the first melting domain (Helms,1990). Placing two samples side-by-side on the gel and allowing them to denature together, researchers can easily see even the smallest differences in two samples or fragments of DNA. Pulse Field GE used for the separation of large DNA molecules by changing periodically the electric field from a gel matrix. In a standard gel, DNA molecules bigger than 15 Kb move together regardless of their size. PFGE is a variation that introduces alternating voltage gradient to improve the resolution of larger molecules. The voltage is periodically switched among three directions: (1)To the central axis of the gel, (2,3) at an angle of 60 degrees from both sides. The result is that DNA does not move in a straight line through the gel but in an "net forward" migration pattern. Pulse Field GE END OF THIS TOPIC References Schleif R. Genetics and Molecular Biology, Second Edition. The Johns Hopkins University Press. Weaver, R.F. 2001. Molecular Biology. McGraw-Hill.