Molecular Diagnostic Techniques Chapter 12 PDF

6/27/2024 Molecular Diagnostic Techniques...

6/27/2024 Molecular Diagnostic Techniques Chapter 12 Preamble ° PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. PowerPoints DO NOT cover the details needed for the Unit exam ° Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES ° Unit Objectives are your study guide (not this PowerPoint) ° Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! 1  6/27/2024 Chapter Overview Characteristics of DNA and RNA Electrophoresis Strand cleavage methods Hybridization methods Amplification methods DNA sequencing Molecular Diagnostic Techniques Performed to gain information to aid in diagnosis, prognosis, and monitoring of disease, as well as treatment decisions. Detect specific nucleic acid sequences in microorganisms or cells before antibody detection is possible. 2  6/27/2024 Deoxyribonucleic Acid (DNA) ° Nucleic acid that carries the primary genetic information within chromosomes ° Contains the sugar deoxyribose ° Purine bases: adenine, guanine ° Pyrimidine bases: cytosine, thymine Ribonucleic Acid (RNA) ° Helps convert the genetic information encoded within DNA into proteins ° Contains the sugar ribose ° Purine bases: adenine, guanine ° Pyrimidine bases: cytosine, uracil ° Types of RNA: Messenger RNA (mRNA) Transfer RNA (tRNA) Ribosomal RNA (rRNA) Noncoding RNAs 3  6/27/2024 Nucleotides ° Units that make up DNA or RNA ° Composed of: A deoxyribose sugar (DNA) or ribose sugar (RNA) A nitrogen base A phosphate group DNA and RNA Polymers 4  6/27/2024 DNA Replication ° The two DNA strands separate. ° Each strand is a template for synthesis of a complementary strand. ° DNA polymerase is needed for synthesis. ° Each new strand is synthesized in the 5’ to 3’ direction as the original double strand unwinds. ° Two identical double-stranded molecules result. RNA Synthesis Can start de novo without a primer. Is catalyzed by RNA polymerase. Occurs throughout the cell cycle. mRNA codes for the amino acid sequence of a protein. 5  6/27/2024 Protein Synthesis and Translation Messenger RNA (mRNA) is transcribed from DNA using RNA polymerase. The mRNA delivers the information to ribosomes, where protein synthesis takes place. As each amino acid is added, the peptide chain continues to grow. This translation process is accomplished with the help of tRNA, which brings in individual amino acids. DNA Sequence Changes Mutations and variants Changes are in the nucleotide sequence of DNA. Some are associated with disease. Variants are inherited, whereas mutations are spontaneous changes in somatic cells. Polymorphisms Alterations in DNA or protein sequences shared by at least 2% of the population The MHC is a highly polymorphic region in the human genome. 6  6/27/2024 Electrophoresis ° Laboratory procedure commonly used in molecular diagnostics ° Involves movement of particles under the force of an electrical current. ° Electrophoresis of nucleic acids commonly takes place in a semisolid matrix called a gel. ° Nucleic acids are negatively charged and migrate toward the positive pole (anode). ° Smaller nucleic acid chains migrate faster than large chains. Capillary Electrophoresis A more sensitive, semi- automated method that separates particles in a gas, liquid, or gel DNA chains carry a fluorescent label that is excited by a laser to produce peaks of fluorescence. 7  6/27/2024 Molecular Analysis Four Main approaches to nucleic acid analysis: Nucleic acid tests are designed to detect Strand Cleavage methods changes (mutation and polymorphisms) in the Hybridization methods DNA sequence. Amplification methods Sequencing methods Molecular Analysis: Strand Cleavage Methods ° Use restriction endonuclease enzymes to cleave DNA at specific locations. ° Separate cleaved fragments by size and charge using electro-phoresis, revealing potential variations. 8  6/27/2024 Strand Cleavage Methods (continued) Used to investigate small genomes, such as those of microorganisms or plasmids. Detect mutations and polymorphisms. CRISPR-Cas9 is a newer method that uses an enzyme to alter DNA at user-defined locations identified by a small guide RNA molecule. It has been used for DNA analysis, gene therapy, and genome editing. Molecular Analysis: Hybridization Methods Hybridization involves the binding of two complementary nucleic acids, most notably a template and a probe Hybridization methods are used on large, complex genomes. Examples: Southern blot, Microarray, Bead arrays and In situ hybridization 9  6/27/2024 Microarrays Allow for multiple targets or samples to be analyzed simultaneously. The test sample is labeled with a fluorescent dye and added to a glass slide containing thousands of specific unlabeled probes. Fluorescent spots appear where complementary binding occurs. Types of Microarrays Comparative Detect amplifications or deletions in DNA. genomic arrays RNA expression Detect genes that have been actively transcribed into arrays mRNA. Detect single nucleotide differences in test samples, some SNP arrays of which may be associated with a particular disease. 10  6/27/2024 Bead Arrays ° Used for multiplex detection of proteins and nucleic acids. Tissue typing Respiratory virus panels ° Beads may have antibodies or single-stranded oligonucleotides attached. In Situ Hybridization Fluorescence in situ hybridization (FISH) Uses fluorescent labeled probes to detect specific DNA sequences. Labeled probes hybridize to Detects chromosome abnormalities (e.g., tissues or cells on glass slides. microdeletions, gene amplifications). Immunohistochemistry Uses enzyme-labeled antibodies to identify protein targets. 11  6/27/2024 Target Amplification: Polymerase Chain Reaction (PCR) 1. Denaturation DNA is separated into single strands by heat (~95oC). 2. Annealing Primers attach (~60oC). 3. Extension Complementary nucleotides are added to 3’ end (~70oC). Amplification Methods: PCR Steps Target amplification ° Amplification of PCR products is an exponential process in which, under optimal conditions, each cycle results in a doubling of product ° In this manner, a target sequence present at fewer than 100 copies can be detected. 12  6/27/2024 Amplification Methods: PCR Measurements The resulting DNA fragments, referred to as amplicons, are detected using various methods. Initially, amplicons were detected by visualization on agarose gels. Currently, for most clinical applications, amplicons are detected using Target labeled nucleic acid probes. amplification PCR Video: https://www.youtube.com/watch?v=2KoLnIwoZKU PCR Modifications Reverse transcriptase Quantitative PCR Digital droplet PCR PCR (RT-PCR) (qPCR) Amplifies cDNA Determines the Provides absolute made from an RNA amount of a specific quantification of template (e.g., HIV, sequence in the PCR product. HCV). original sample by incorporation of fluorescent probes as the PCR product is made. 13  6/27/2024 Transcription-Based Amplification (TMA) ° RNA is the target as well as the primary product. ° Product detected by chemiluminescence. ° Useful in testing for RNA viruses (e.g., HIV). Strand Displacement Amplification (SDA) ° A probe amplification method 14  6/27/2024 Branched DNA (bDNA) A signal amplification Has been method that used to uses multiple detect certain reporter viruses. molecules. DNA Sequencing The most definitive method of Methods include Sanger Direct determination of the identifying genetic mutations or polymorphisms, especially sequencing, pyrosequencing, order of nucleotides in a DNA when changes affect only one and next-generation chain. sequencing (NGS). or two nucleotides. 15  6/27/2024 Sanger Sequencing Also known as chain termination sequencing Uses all components of PCR plus fluorescent-labeled dideoxynucleotides (ddNTPs) to synthesize DNA complementary to the target. When the correct ddNTP is added, DNA synthesis stops and fragments of varying sizes are produced. Results are read by gel or capillary electrophoresis. Pyrosequencing ° Based on the generation of light when nucleotides are added to a growing strand of DNA 16  6/27/2024 Next-Generation Sequencing (NGS) ° Can sequence large numbers of templates simultaneously (massively parallel sequencing). NGS by Reversible Dye Terminator Sequencing ° Using bioinformatics, the short sequences are assembled to determine the complete sequence, which is compared to known databases. ° Applications include identification of genetic variations in inherited diseases and tumor cells, HLA allele typing, and analysis of mixed microbial populations. Summary Nucleotides of DNA contain a deoxyribose sugar, a phosphate group, and a base of adenine, guanine, thymine, or cytosine. RNA has nucleotides containing a ribose sugar bonded to a similar nitrogen base as in DNA, but uracil is used instead of thymine. DNA is double stranded and arranged in a double helix, whereas RNA is typically single stranded. 17  6/27/2024 Summary (continued_1) In a DNA molecule, adenine pairs with thymine, and guanine pairs with cytosine. When a DNA molecule replicates, the two daughter strands separate; each is a template for a newly synthesized complementary strand. Mutations and polymorphisms are changes in nucleotide sequences that may affect specific protein sequences. Summary (continued_2) Hybridization is the very specific binding of two complementary DNA strands or a DNA and an RNA strand. A labeled probe is often used to detect an unknown nucleic acid sequence. Hybridization techniques include microarray technology and fluorescent in situ hybridization (FISH). 18  6/27/2024 Summary (continued_3) Amplification techniques involve copying of a specific nucleic acid sequence to obtain enough material for laboratory identification. Amplification methods include PCR, RT-PCR, qPCR, and digital PCR. All these methods amplify the target DNA. In transcription-mediated amplification (TMA), a cDNA copy made of the original RNA is used to produce millions of RNA copies. Summary (continued_4) Strand displacement amplification (SDA) involves amplification of a probe rather than the original target DNA. Branched DNA represents a signal amplification method; multiple probes attach to the original target-sequence DNA. 19  6/27/2024 Postamble ° READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES. ° USE THE UNIT OBJECTIVES AS A STUDY GUIDE ° All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives 20