Methods in Studying Genetics PDF Lecture Notes

Wrap-up of the previous sessions…. Methods in Studying Genetics Maribel D. Ganeb, Ph.D, LPT  Highlights üKaryotyping üChromosomal banding üPolymerase chain reaction üGel electrophoresis üWhole genome sequencing üGene cloning  Karyotype ü A karyotype is the complete set of...

Wrap-up of the previous sessions…. Methods in Studying Genetics Maribel D. Ganeb, Ph.D, LPT  Highlights üKaryotyping üChromosomal banding üPolymerase chain reaction üGel electrophoresis üWhole genome sequencing üGene cloning  Karyotype ü A karyotype is the complete set of chromosomes in a specie. It describes the number of chromosomes, and what they look like under a light microscope. ü It is a photographic representation of a stained metaphase spread in which the chromosomes are arranged in order of decreasing length.  Karyotype Specimens that contain spontaneously proliferating cells include bone marrow, lymph nodes, solid tumors, and chorionic villi Peripheral blood lymphocytes, tissue biopsies, and amniotic fluid samples are routinely cultured to obtain dividing cells; lymphocytes usually require the addition of a mitotic stimulant. The choice of specimen for chromosome analysis depends on clinical indications and whether the diagnosis is prenatal or postnatal  CHROMOSOME STAINING AND BANDING A chromosome band is a part of chromosome that is clearly distinguishable from its adjacent segments comprised of alternating light and dark stripes.  CHROMOSOME STAINING AND BANDING Banding and staining techniques can be divided into two broad categories: those that produce specific alternating bands along the length of each entire chromosome and those that stain only a specific region of some or all chromosomes Techniques that Stain Selective Chromosome Regions 1. C-Banding (Constitutive Heterochromatin Banding) − Useful for determining the presence of dicentric and pseudodicentric chromosomes, and for studying marker chromosomes and polymorphic variants. − With CBG banding (C-bands, by barium hydroxide, using Giemsa), the DNA is selectively depurinated and denatured by barium hydroxide, and the fragments are washed away by incubation in a warm salt solution.  Techniques that Stain Selective Chromosome Regions 1. − selectively stain the constitutive heterochromatin around the centromeres, the areas of inherited polymorphisms present on chromosomes 1, 9, and 16, and the distal long arm of the Y chromosome. Normal Chromosome Dicentric  Techniques that Stain Selective Chromosome Regions 2. T-Banding (Telomere Banding) ­ A harsher treatment of the chromosomes diminishes staining except at the heat-resistant telomeres. 3. NOR Staining (Silver Staining for Nucleolar Organizer Regions) ­ Nucleolar organizer regions (NORs) located on the satellite stalks of the acrocentric chromosomes. ­ Silver nitrate is used to stain the regions that contain genes for ribosomal RNA Cytogenetics  Cytogenetics focuses on the microscopic examination of genetic components of the cell, including chromosomes, genes, and gene products. Biochemical methods are applied to the main chemical compounds of genetics—notably DNA, RNA, and protein. Biochemical techniques are used to determine the activities of genes within cells and to analyze substrates and products of gene- controlled reactions. POLYMERASE CHAIN REACTION (PCR)  WHAT IS PCR? called "molecular photocopying," used to "amplify" - copy - small segments of DNA. is a novel molecular technique involving in vitro enzymatic replication of defined DNA sequences used to diagnose diseases, identify bacteria and viruses, match criminals to crime scenes, and biotechnology.  How does PCR work? In a typical PCR experiment, the target DNA is mixed with Taq polymerase, the two oligonucleotide The PCR reaction has 3 primers, and a supply of deoxyribonucleoside triphosphates (dNTPs) in buffer. steps: denaturation, annealing and extension  Common PCR Master Mix Component Volume Final concentration 10X PCR Buffer 5 µL 1X dNTPs (10 mM) 1 µL 200 µM Forward primer 1 - 2 µL 50 - 100 pmol Reverse primer 1 - 2 µL 50 - 100 pmol 2.5 - 5 U Taq DNA polymerase (5U/µL) 0.5 - 1 µL Template DNA 1 - 5 µL 1 - 200 ng MgCl2 (50 mM) 1 µL 1 mM PCR Water Variable Add to q.s. to 50 µL Total volume 50 µL https://www.sigmaaldrich.com/life-science/molecular-biology/pcr/pcr-master-mix.html?gclid= EAIaIQobChMIxKiLl4G96gIVGLaWCh2ITgT5EAAYASAAEgI2rfD_BwE PCR Protocol 1. Denaturation of dsDNA template – thermal denaturation of dsDNA at 94℃ 2. Annealing of two oligonucleotide primers – temp. is decreased to 50-60℃ Denature at 94℃ Anneal at 50-60℃ which allows primers to attach to complementary sequences 3. Polymerase extension of DNA Extend at 72- 74℃ molecules – temp. is raised at 72-74℃ just below the optimum of Taq polymerase Standard 3-Step PCR Cycling Cycle step Temperature Time Number of Cycles Initial 94 °C to 98 °C 1 minute 1 Denaturation Denaturation 94 °C 5 °C below 10 to 60 seconds 30 25-35 Annealing Tm 50 °C to 60 °C seconds Amplicon and DNA polymerase dependent Final Extension 70 °C to 80 °C 5 minutes 1 Hold* 4 °C ∞ 1 Most thermal cyclers can pause at 4°C indefinitely at the end of the cycles *Lorenz, T. (2012)  Modified Nucleic Acid Amplification Techniques  -developed to amplify RNA targets Reverse- Transcriptase -RNA molecule via reverse transcriptase enzyme is converted to cDNA molecule Polymerase and then utilized as template sequence for following PCR reaction Chain Reaction Application: detect RNA expression Nested Polymerase Chain Reaction Multiplex Polymerase Chain Reaction Digital Polymerase Chain Reaction Quantitative PCR (qPCR) or real-time PCR - In real-time PCR, the amplification reaction is monitored in “real time.” - Monitor the amplification of a targeted DNA molecule during the PCR with use fluorescent dyes or fluorophore-containing DNA probes such as Taq man An example of PCR amplification plot, where the threshold cycle (𝐶! ) is the cycle number at which the fluorescence crosses the amplification threshold.  GEL ELECTROPHORESIS Electrophoresis ü A method of separating electrically charged substances in a mixture ü A sample of the mixture is placed on a supporting medium, to which an electrical field is applied General Principle Ø Each charged substance migrates toward Anode Cathode cathode or the anode at a speed that + depends on its net charge, size and shape. Ø When an electrical field is applied to a solution, solute molecules with a net positive charge migrate toward the cathode, - and molecules with a net negative charge move toward the anode. Ø Electrostatic attraction causes biomolecules to migrate towards the electrodes of opposite charge Father of Electrophoresis Arne Tiselius did pioneer work on moving boundary electrophoresis (1930) and later developed a zone method for the purification of biomolecules.  Electrophoresis : 3 PARTS Buffer + + + vSupporting medium: Electric vVoltage: power HIGH Field in Gel Matrix in which the VOLTAGE supply biomolecules separation takes place - - - Buffer vBuffer system : Conduct electricity by running buffer  A type of electrophoresis in which supporting medium is a gel Separation is brought about through Gel molecular sieving technique, based on molecular size of the substances Electrophoresis Gel material acts as a “molecular sieve” The technique is widely used for separating proteins and nucleic acids Gel Electrophoresis : 2 Methods Horizontal Gel Electrophoresis Supporting Medium Vertical Gel Supporting Medium Electroph oresis Buffer System Voltage or Power Supply Buffer System  Types of Gels Agarose Polyacrylamide Gel Polysaccharide extracted from seaweed Cross-linked polymer of acrylamide Gel casted horizontally Gel casted vertically Non-toxic Potent neuro-toxic Separate large molecules Separate small molecules Commonly used for DNA separations Used for DNA or protein separations Staining can be done before or pouring the gel Staining can be done after pouring the gel Method for Electrophoresis Run gel at constant voltage View DNA on UV light box and show until band separation occurs results  a photograph of an actual agarose gel This is a photograph of an actual agarose gel that has been run with samples of DNA. For each question, write the letter from the item in the gel that best matches the word or concept that is being described. Amplicon band: DNA Ladder: Well: Which band contains smaller DNA fragments, F or G? Where would the amplicon be loaded? WHOLE GENOME SEQUENCING  WHAT IS WHOLE GENOME SEQUENCING? Genome sequencing involves finding out the whole sequence of a person’s DNA. laboratory procedure that determines the order of bases in the genome of an organism in one process.  WHOLE GENOME SEQUENCING: DISCOVERY Sanger and his colleagues invented chain termination sequencing I 1970 Fragments of 100-1000 base pair In 1979 SHOTGUN SEQUENCING TECHNIQUE was developed Large size of fragments Overlapping of discovered fragments to detect whole genome sequences.  How does whole genome sequencing work? Scientists conduct whole genome sequencing by following these four main steps: DNA shearing: Scientists begin by using molecular scissors to cut the DNA, which is composed of millions of bases: A’s, C’s, T’s and G’s, into pieces that are small enough for the sequencing machine to read. DNA bar-coding: Scientists add small pieces of DNA tags, or bar codes, to identify which piece of sheared DNA belongs to which bacteria. This is similar to how a bar code identifies a product at a grocery store.  How does whole genome sequencing work? Whole genome sequencing: The bar-coded DNA from multiple bacteria are combined and put in the whole genome sequencer. The sequencer identifies the A’s, C’s, T’s, and G’s, or bases, that make up each bacterial sequence. The sequencer uses the bar code to keep track of which bases belong to which bacteria. Data analysis: Scientists use computer analysis tools to compare bacterial sequences and identify differences. The number of differences can tell the scientists how closely related the bacteria are, and how likely it is that they are part of the same outbreak.  WHOLE GENOME SEQUENCING: ADVANTAGES Creating personalized plans to treat disease may be possible based not only on the mutant genes causing a disease, but also other genes in the patient’s genome. Genotyping cancer cells and understanding what genes are mis regulated allows physicians to select the best chemotherapy and potentially expose the patient to less toxic treatment since the therapy is tailored. WHOLE GENOME SEQUENCING: ADVANTAGES Previously unknown genes may be identified as contributing to a disease state. Traditional genetic testing looks only at the common “troublemaker” genes. Lifestyle or environmental changes that can mediate the effects of genetic predisposition may be identified and then moderated. WHOLE GENOME SEQUENCING: DISADVANTAGES The role of most of the genes in the human genome is still unknown or incompletely understood. Most physicians are not trained in how to interpret genomic data. An individual’s genome may contain information that they DON’T want to know. The volume of information contained in a genome sequence is vast. GENE CLONING GENE CLONING GENE CLONING Thank you for Listening! Associate Professor Maribel D. Ganeb