2023 Molecular Biology and Diagnostics Main Module PDF

Molecular Biology and Diagnostics Main Module for Medical Technologists (2023 Edition) Prepared by: Prof. Joshua Angelo Hermida Mandanas International Researcher/Scientist and Educator of Molecular Biology, Tropical Medicine and Biochemistry...

Molecular Biology and Diagnostics Main Module for Medical Technologists (2023 Edition) Prepared by: Prof. Joshua Angelo Hermida Mandanas International Researcher/Scientist and Educator of Molecular Biology, Tropical Medicine and Biochemistry National Medical Technology Board Reviewer and Book Author Citation: “Molecular Biology and Diagnostics Main Module for Medical Technologists (2023 Edition). 052023. Philippines” Do not upload in any social media platforms and distribute to other institutions. This is not for sale. Topic: Introduction to Molecular Biology and Related Fields  Molecular Biology: deals with the composition, structure and functionality of nucleic acids (genes to proteins). Applications in Biotechnology, Research and Development  Biochemistry: study of the chemical composition and properties of living matter, biochemical processes underlying life and translational research. (Carbs/Lipids/Nucleotide/Proteins)  Genetics: study of genes, functions and factors related to all aspects of genes/ heredity  Cell biology: study of the structure, function and regulations of the cell  Quantum mechanics/Biophysics: study of the smallest units and physical properties of matter, from molecules to atoms and subatomic particles Overview of the central dogma of Molecular Biology-For each component, identify the possible applications of Molecular Biology… Examples of Molecular biology applications… Others: Development of biomarkers, compounds/vaccines against infectious and/or degenerative diseases Page 1 of 42 MAIA Professional Development Center August MTLE 2023 Topic: Review of Medical Technology Chemistries  General/Inorganic Chemistry/Atomic Science: *Light (wave-like-wavelength, frequency; particle-photon) *Mass number: # of protons and neutrons; Atomic number: # of electrons *Isotopes are members of a family of an element that have the same number of protons but different numbers of neutrons Page 2 of 42 MAIA Professional Development Center August MTLE 2023  Analytical Chemistry: Note: Covalent bond [(sharing of electrons-e.g. polar (water), nonpolar(hydrocarbons, diatomic)]; Noncovalent bond (electromagnetic interactions- e.g. van der waals and hydrogen bond) Number of bonds in a covalent bond… Page 3 of 42 MAIA Professional Development Center August MTLE 2023  Organic Chemistry Acid hydrolysis of glycosidic bond….> cAMP and GTP  Types of DNA (note-Denatured DNA is hyperchromic): Types of RNA: Molecular interaction of DNA bases Page 9 of 42 MAIA Professional Development Center August MTLE 2023  Franklin and Wilkins (1950s): DNA X-ray diffraction-helical nature; Watson and Crick (1953): 3D model of DNA Hershey-Chase Experiment (DNA, 1950s) Gene as a transcriptional unit Page 10 of 42 MAIA Professional Development Center August MTLE 2023  (arms-anticodon, AA, D, TΨC arm, extras)-cloverleaf-like Interaction of mRNA, tRNA and ribosomes during translation Page 11 of 42 MAIA Professional Development Center August MTLE 2023 Topic: Chromosomal and Cell biology Story of the chromosomal packaging Overview of human genome gene-related sequences (30%); 70% (extragenic) Page 12 of 42 MAIA Professional Development Center August MTLE 2023 DNA homologous recombination: process by which pieces of DNA are broken and recombined to produce new combinations of alleles. This recombination process creates genetic diversity at the level of genes that reflects differences in the DNA sequences of different organisms. For prokaryotes (repair); eukaryotes (genetic diversity)  Mutations: Missense (AA->diff.AA); Nonsense (stop codon); silent (no AA change)  Aneuploidy-extra or missing chrom.: Down syndrome (Trisomy 21); Patau (Trisomy 13); Edwards (Trisomy 18) Patterns of inheritance (Mendelian, single-gene diseases): a. Autosomal dominant- child inherits one copy of a mutated (changed) gene from one parent. (e.g. Huntington’s disease, familial hypercholesterolemia) Page 13 of 42 MAIA Professional Development Center August MTLE 2023 b. Autosomal Recessive- two copies of the mutated gene (one from each parent) are required to cause the disorder (e.g. Tay- sachs disease, sickle cell anemia, cystic fibrosis, phenylketonuria) c. X-linked dominant- a single copy of the mutation is enough to cause the disease in both males (who have one X chromosome) and females (who have two X chromosomes). vitamin D-resistant rickets d. X-linked recessive- a male carrying such a mutation will be affected, because he carries only one X chromosome. A female carrying a mutation in one gene, with a normal gene on the other X chromosome, is generally unaffected. (e.g. Hemophilia A, Duchenne muscular dystrophy) DNA repair during a mutation Page 14 of 42 MAIA Professional Development Center August MTLE 2023 Topic: Molecular Theories (Central Dogma and updates) Fill-in this table! Replication Transcription Translation Initiation Elongation Termination I. DNA Replication Replication begins when origin-binding proteins bind to the origin of replication on the DNA. Then the unwinding of the double helix proceeds by way of the enzyme, helicase, creating a replication fork. The replication fork is Y- shaped and is where the leading and lagging strands are formed Page 15 of 42 MAIA Professional Development Center August MTLE 2023 Important components of DNA replication: a. Biochemical enzymes (helicase, topoisomerase/gyrase, primase, DNA polymerase, ligase) b. Building blocks-dATP, dCTP, dGTP, dTTP c. Forward and Reverse Primers, chromosomal DNA templates d. Origin sites- prokaryotes (GATCTNTTNTTTT, TTATCCACA), eukaryotes (many) DNA polymerase III subunits (top) and summary of molecular action (right) DNA polymerase starts synthesizing the new strand from 5’ to 3’ direction, but requires a short RNA sequence, approximately 10 nucleotides in length, as a primer to begin the process. An RNA primer is therefore generated with the help of RNA primase enzyme, to initiate placing RNA bases complementary to the template strand bases. On the leading strand, the DNA polymerase continues in constant motion while the lagging strand has to be copied in short segments in the opposite direction since DNA polymerase only codes in 5' to 3' direction. These short repetitive lagging fragments are known as the "Okazaki fragments” which are connected by DNA ligase.  DNA polymerase: prokaryotes (pol I-Klenow fragment, II, III); eukaryotes (a,d-PCNA,e)  Plasmid: extrachromosomal circular DNA Page 16 of 42 MAIA Professional Development Center August MTLE 2023  Molecular actions of DNA helicase, topoisomerase and binding proteins Molecular logic of consensus sequence Summary of the eukaryotic cell cycle Prokaryotic DNA replication Page 17 of 42 MAIA Professional Development Center August MTLE 2023 Biochemical action of DNA ligase…> Biochemical action of DNA polymerase Page 18 of 42 MAIA Professional Development Center August MTLE 2023  Actions of DNA topoisomerase/gyrase (left-biochemical; right-overview)  Answer this question: where does DNA replication happens in prokaryotes?in eukaryotes?  Medical impact: Acyclovir (acts as chain terminator); Quinolones (inhibits DNA gyrase) II. Translation a. Biochemical enzymes (RNA polymerase, topoisomerase/gyrase) b. Building blocks-ATP, CTP, GTP, UTP c. Template DNA d. Important sequences: promoter, enhancer and operator sequences While prokaryotes have a single RNA polymerase, eukaryotes possess three RNA polymerases. RNA synthesis requires RNA polymerase I for ribosomal RNA, RNA polymerase II for mRNA and microRNA, and RNA polymerase III for tRNA and other small RNAs. Transcription is not as accurate as replication and produces more errors. Summary of the molecular functions of enhancer and operator sequences Page 19 of 42 MAIA Professional Development Center August MTLE 2023  The molecular process of mRNA transcription Answer this question: The mRNA sequence identified by a Registered Medical Technologist is found to be:(5’)- U U U A A A G G G C C -(3’); what is the coding sequence?_______________________ Prokaryotic Eukaryotic RNA polymerase RNA Pol Pol II Promoter sequences -10: 5’-TATAAT-3’ Hogness box -35: 5’-TTGACA-3’ -UP element:5’- -40 to -60 -3’ Page 20 of 42 MAIA Professional Development Center August MTLE 2023  Promoter sequences are important during transcription Shine-Dalgarno sequences  Medical Impact: Acridine (DNA intercalator); Rifampicin (binds to bacterial pol)  DNA and RNA polymerase have the similar molecular functionality III. RNA Processing Once synthesized, the RNA transcript from the nucleus to the cytoplasm where it will be translated to protein. The single- stranded RNA receives a 5' capping by 7-methylguanosine as well as the addition of poly-A-tail on the 3' end. During these modifications, eukaryotic cells also undergo intron splicing, leaving only the required bases for translation, known as exons. Page 21 of 42 MAIA Professional Development Center August MTLE 2023  RNA processing core steps: capping, splicing and polyadenylation a. Capping (head): protection against ribonucleases,promotes binding to ribosomes/proteins b. Splicing: intron removal c. Polyadenylation (tail): protection against enzymes, promotion of protein binding Introduction to Protein Translation  Nomenclature: tRNAala: specific for alanine; Ala-tRNAala: aminoacylated or alanine has already charged to its respective tRNA  Ribosome movement: 5’->3’; tRNA addition: 3’-> 5’  Errors of translation:1 per 104 amino acids incorporated in tRNA Summary of amino acid nomenclature (left) and codon library (right) Page 22 of 42 MAIA Professional Development Center August MTLE 2023 IV. Translation Part one: Amino acid activation by aminoacyl tRNA-synthetase Part two: Charging of amino-acyl product also by aminoacyl tRNA- synthetase The initial step of the initiation stage is the binding of a specific initiator methionyl tRNA and the mRNA to the small ribosomal subunit. The large ribosomal subunit then joins the complex, forming a functional ribosome on which elongation of the polypeptide chain proceeds (large SU contains peptidyl transferase) Elongation of the polypeptide chain continues until a stop codon (UAA, UAG, or UGA) is translocated into the A site of the ribosome. Cells do not contain tRNAs with anticodons complementary to these termination signals; instead, they have release factors that recognize the signals and terminate protein synthesis Medical Impact: Chloramphenicol (inhibits peptidyl transferase); Tetracycline (inhibits 30 S A site); Cycloheximide (fungal/eukaryotic peptidyl transferase) Page 23 of 42 MAIA Professional Development Center August MTLE 2023 V. Special mechanisms a. RNA-dependent-DNA polymerase a.1. Reverse transcriptase This enzyme has the polymerase activity and ribonuclease H (RNase H) activity both in p66, and is responsible for the reverse transcription of retroviral single-stranded RNA into double-stranded DNA. p51 provides structural support. Nucleoside RT inhibitor (NRTI)-Zidovudine NNRTI-Efavirenz, Nevirapine a.2. Telomerase Towards the completion of replication, the newly synthesized lagging strand is shorter with a 3’- overhang. This is thought to be due to the degradation of RNA at the end of the chromosome or inability of primase to lay down a primer at the end. To prevent the loss of genes during successive replications, telomeres are added at the ends of the chromosomes by an enzyme called telomerase. Without it, the chromosomes will gradually shorten with each replication and the cell undergoes premature senescence. b. RNA-dependent-RNA polymerase  Found in RNA viruses (clinical correlation, Remdesivir-chain termination) Page 24 of 42 MAIA Professional Development Center August MTLE 2023  Viral Genomic Replication (RNA viruses) VI. Summary of post-translationally modified proteins in humans Molecular levels of Protein Structure- Primary structure (AA sequence)->Seconday (helix, pleated sheets) ->Tertiary (covalent and noncovalent)-> Quaternary (Arrangement with subunits) Page 25 of 42 MAIA Professional Development Center August MTLE 2023 Topic: Quality control in the Molecular Biology Laboratory  Differential centrifugation: tissue homogenate-> low speed (1000 g)-> medium speed (20,000 g)-> high-speed (80,000 g)  BSL-1(routine Molbio techniques); BSL-2(sequencing); BSL-3 (viral culture)  Molecular testing anticoagulant of choice: EDTA if cytogenetics (heparin) Isopycnic (Density Gradient Centrifugation) Basics of nucleotide isolation For RNA- (treat with dithiothreitol- sulphide bond reduction, or diethylpyrocarbonate- converts amines to esters). RNA needs enrichment through synthetic probes/beads Answer this! What are the basic steps in DNA preparation and isolation? Topic: Amino acid Technologies I.Enzymatic degradation Proteolytic enzyme Target Position Pepsin Aromatic amino acids (AA) Amino terminal Trypsin Basic/positively charged AA Carboxy terminal Carboxypeptidase B Page 26 of 42 MAIA Professional Development Center August MTLE 2023 II. Sequencing technologies: a. Edman degradation: label and cleave the peptide from N-terminal without disrupting the peptide bonds between other amino acid residues. b. Sanger degradation III. Types of purification techniques a. Affinity chromatography: separation method based on a specific binding interaction between an immobilized ligand and its binding partner (e.g. enzyme-subtrate/inhibitor; antibody-antigen) b. Ion exchange chromatography- separation of charged molecules Page 27 of 42 MAIA Professional Development Center August MTLE 2023 c. Gel (size-exclusion) chromatography  Agarose-based matrix: Sepharose 6B (10-4000 kDa), Sepharose 4B (60- 20,000 kDa)  Dextran-based: Sephadex G-10 (0- 0.7 kDa), Sephadex G-100 (4-150 kDa) d. High performance liquid chromatography- column chromatography that pumps at high pressure a sample (analyte) dissolved in a solvent (mobile phase) through a column with an immobilized chromatographic packing material (stationary phase). The properties of the sample and the solvent, as well as the nature of the stationary phase, determine the retention time of the analytes, or how fast they pass through the column. e. Mass spectrometry- an analytical tool useful for measuring the mass-to- charge ratio (m/z) of one or more molecules present in a sample. Ionized sample molecules are separated according to their mass to charge ratios (m/z) by the application of electric and/or magnetic fields. Topic: Electrophoresis and blotting techniques I. Electrophoresis: laboratory technique used to separate DNA, RNA or protein molecules based on their size and electrical charge. An electric current is used to move the molecules through a gel/matrix Gels used: DNA (Agarose-made from galactopyranose with its anhydrous form): 0.6% for 5000-60,000 DNA bp; 0.8% for 800-1000 bp; 1% for 400-8000 bp and 2% for up to 3000 bp Protein (Polyacrylamide)-made from acrylamide and bis-form (reaction catalysed by ammonium persulfate and Tetramethylethylenediamine Page 28 of 42 MAIA Professional Development Center August MTLE 2023  Qualitative analysis of polyacrylamide gel result II. Summary of blotting techniques Important terminologies: Hybridization- exploits the ability of complementary sequences in single- stranded DNAs or RNAs to pair with each other to form a double helix. Hybridization can take place between two complimentary DNA sequences, between a single-stranded DNA and a complementary RNA, or between two RNA sequences (application-PCR, FISH) Molecular principle of fluorescence in situ hybridization (FISH) Chromosomal techniques: a. Karyotyping (metaphase chromosomes) b. FISH (specific probes c.Banding/staining : C (centromere); G (heterochromatin),R( euchromatin) Page 29 of 42 MAIA Professional Development Center August MTLE 2023  Blotting-transfer into nitrocellulose membrane (capillary, vacuum) a. Southern blot- transferring DNA molecules from an agarose gel to a surface such as a positively charged nylon transfer membrane b. Northern blot- uses denaturing gel to separate RNA according to the size. The RNA is then transferred to a nylon membrane while keeping the same distribution in the gel. After fixing the RNA to the membrane, labeled probe complementary to the gene of interest is then added to hybridize to the immobilized RNA. c. Western blot- mixture of proteins are separated based on molecular weight, and thus by type, through gel electrophoresis. These results are then transferred to a membrane producing a band for each protein III. Special mention: Pulsed-gel field electrophoresis: separation of large DNA molecules (entire genomic DNA) after digesting it with unique restriction enzymes and applying to a gel matrix under the electric field that periodically changes direction. It is considered the “gold standard” of bacterial typing. Page 30 of 42 MAIA Professional Development Center August MTLE 2023 Topic: DNA techniques I. Recombinant DNA technology  it involves molecular cloning of the DNA insert being linked to vector DNA sequences. Large quantities of the inserted DNA can be obtained if the recombinant molecule is allowed to replicate in an appropriate host. II. Restriction endonucleases (biotechnology, bioengineering)  enzymes that cut DNA at specific sequences. Naturally found in bacteria to defend against viral pathogens Topic: Target Amplification I. Maxam –Gilbert  A+G (acid hydrolysis); G (dimethylsulfate treatment); C (piperazine + hydrazine); C+T (NaCl) Page 31 of 42 MAIA Professional Development Center August MTLE 2023 II. Sanger sequencing  DNA polymerase adds dNTPs to a growing DNA strand by catalyzing the formation of a phosphodiester bond between the free 3’-OH group of the last nucleotide and the 5’- phosphate of the next. The scientist mixes a low ratio of chain-terminating (dideoxy) ddNTPs in with the normal dNTPs in the PCR reaction. III. Polymerase Chain Reaction  The theoretical process was outlined by Keppe and coworkers in 1971  It was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus corporation in 1985  PCR workflow: 1. Pre-analytical: sample collection/receipt, transport conditions, primer designing (use the formula [2 (A + T) + 4 (G + C)] 2.Analytical: Pre-PCR(Nucleotide extraction, Mastermix prep); PCR(Loading in machine); Post-PCR (Electrophoresis-note electrophoretic gel products can be digested for PCR) *Mastermix-buffer (pH 8-9.5), dNTPs, forward and reverse primer, salt (MgCl2), Taqman polymerase/thermostable DNA polymerase (Taq,Thermus aquaticus), PCR-grade water, template (add this to controls except sample tube), fluorescent label (e.g. Taqman system/520nm, SYBR green/490 nm, ethidium bromide/590 nm) *Controls- positive, negative, blank, amplification control *Thermocycler machine notes- light source (halogen lamp), Heat source (Peltier metal). Thermocyclers, or polymerase chain reaction (PCR) machines, are laboratory instruments that are used to amplify DNA and RNA samples by the PCR by regulating temperature during cyclical programs Page 32 of 42 MAIA Professional Development Center August MTLE 2023  *Labelling reaction: Taqman system- uses a nucleic acid probe complementary to an internal segment of the target DNA. The probe is labeled with two fluorescent moieties. The emission spectrum of one overlaps the excitation spectrum of the other, resulting in “quenching” of the first fluorophore by the second. 3.Post-analytical: Verification, Releasing & Encoding of result PCR uses the complementary base pairing, double-stranded nature, and melting temperature of DNA molecules. This process involves cycling through 3 sequential rounds of temperature dependent reactions Denaturation begins by heating the reaction to about 95 C, disrupting the hydrogen bonds that hold the two strands of template DNA together. Next, the reaction is reduced to around 50 to 65 C, depending on the physicochemical variables of the primers, enabling annealing of complementary base pairs. Primers, which are added to the solution in excess, bind to the beginning of the 3' end of each template strand and prevent re-hybridization of the template strand with itself. Enzyme-driven DNA replication (elongation) begins by setting the reaction temperature to the amount which optimizes the activity of DNA polymerase, which is around 75 to 80 C. Page 33 of 42 MAIA Professional Development Center August MTLE 2023  Basic steps in PCR Overview of PCR cycles PCR melt curve Page 34 of 42 MAIA Professional Development Center August MTLE 2023 Reverse transcription- Polymerase Chain Reaction PCR Amplification plot REASONS (Faint bands in Agarose) TROUBLESHOOTING GUIDE IN PCR Low starting template concentration Increase template volume Low cycles Increase PCR cycles Long UV exposure Photograph gels as immediately under UV REASONS (Nonspecific bands in Agarose gel) Too much primer concentration Decrease primer concentration Low annealing temperature Increase annealing temperature High number of cycles set Decrease number of cycles Page 35 of 42 MAIA Professional Development Center August MTLE 2023 Topic: Loop mediated isothermal amplification (LAMP)  uses a DNA polymerase with high strand displacement activity and pairs of specially designed inner and outer primers. There is strand displacement in the presence of Bst DNA polymerase under isothermal conditions. Six different primers recognize eight distinct regions on a target gene, with amplification only occurring if all the primers bind and form a product. LAMP is carried out at a constant temperature range of 60–65°C eliminating the need of thermal cycler – makes it a portable tool Topic: Transcription- based amplification isothermal amplification of RNA by reverse transcription and subsequent generation of numerous transcripts by T7 bacteriophage RNA polymerase Topic: Probe Amplification (Ligase chain reaction)  ligation-mediated amplification technique of a target DNA sequence using oligonucleotides and thermostable ligase. It uses biotin-streptavidin detection Page 36 of 42 MAIA Professional Development Center August MTLE 2023 Topic: Signal-based amplification (branched DNA amplification)  uses amplification from signal (e.g. luminescence) to detect nucleotides Topic: Gene-editing technologies [Clustered regularly interspaced palindromic repeats (CRISPR) coupled with CRISPR-associated protein 9 (Cas9), small interfering RNA (siRNA), microRNA (miRNA)] Topic: Next-generation sequencing (principle) Page 37 of 42 MAIA Professional Development Center August MTLE 2023  Additional readings (Molecular and Biochemical Pharmacogenomics) 1. Trastuzumab  monoclonal antibody against human epidermal growth factor receptor 2 (HER2). It binds to an extracellular domain of this receptor and inhibits HER2 homodimerization, thereby preventing HER2-mediated signaling 2. Warfarin  competitively inhibits the vitamin K epoxide reductase complex 1 (VKORC1), an essential enzyme for activating the vitamin K available in the body 3.Clopidogrel  The active metabolite of clopidogrel selectively and irreversibly inhibits the binding of adenosine diphosphate (ADP) to its platelet P2Y12 receptor and the subsequent ADP- mediated activation of the glycoprotein GPIIb/IIIa complex, thereby inhibiting platelet aggregation Page 38 of 42 MAIA Professional Development Center August MTLE 2023 4. Carbamazepine 5. Paracetamol  inhibition of CNS cyclooxygenase (COX) enzyme activities, or prostaglandin synthesis or its active metabolite influencing cannabinoid receptors. Chemical breakdown shown below: 6. BCR/ABL gene rearrangement (Chronic myelogenous leukemia)  this gene produces a chimeric protein (fusion protein) with increased tyrosinase kinase activity which promotes the cells to enter G1, promotes cellular proliferation and suppresses apoptosis. Page 39 of 42 MAIA Professional Development Center August MTLE 2023 7. BCL2 translocation t(14:18)-follicular B-cell lymphoma  t(14;18) joins the BCL-2 gene on chromosome 18 to the immunoglobulin heavy chain gene on chromosome 14, leading to an inhibition of programmed cell death through BCL2 overexpression and consequently, prolonged survival of the affected B cells by production of BCL2 protein which prevents apoptosis 8. BRCA1 and BRCA2 mutations  consists of genes which produces tumor suppressor proteins which promotes cell division and faulty DNA repairs Special feature: Microbial and Viral Genetics A. Plasmid transfer Page 40 of 42 MAIA Professional Development Center August MTLE 2023 B. Bacterial operon (allolactose) C. Yeast hybrid system D. HIV-1 Genome, Peptide profile up to a Virion Page 41 of 42 MAIA Professional Development Center August MTLE 2023 E. SARS-CoV-2 Genome, Peptide Profile and Spike RBD (receptor binding domain)-host ACE2 binding Mandanas (2020) publication. All figures are intended only for educational purposes and not for commercial (profit) MAY MOLECULAR BIOLOGY BRING WONDERS AND HORIZONS UNTO YOUR PATH! Joshua 1:9, Jer 29:11 Always remember you are here in this life for a purpose… You have gifts/talents/skills to bless and help others…A history in the making! Praying for your fruitful and wonderful journeys! Blessings! PROF. JOSHUA ANGELO HERMIDA MANDANAS International Researcher/Scientist and Educator of Molecular Biology, Tropical Medicine and Biochemistry National Medical Technology Board Reviewer and Book Author Advertisement! If you are interested, you may avail/purchase my copyrighted Molecular Biology Book! It contains topics about the Molecular Biology foundations and correlations along with Cellular biology, Molecular diagnostics and Cytogenetics! It is also tagged as the first and recent Comprehensive Molecular Biology Review and Lecture Book for Medical Technologists in the Phils! To avail, purchase and inquire, you may call/text Ms. Grace (publisher) at 09985559130 Low price edition black and white (500 Php); Colored figures edition (950 Php). Deliveries can be made nationwide and outside the Philippines Page 42 of 42 MAIA Professional Development Center August MTLE 2023