Introduction to Molecular Biology and Diagnostics PDF

INTRODUCTION TO MOLECULAR BIOLOGY AND DIAGNOSTICS Mark Laurence A. Manzano, RMT TOPICS OF THE DAY 1 Definition of terms 2 Application and History 3...

INTRODUCTION TO MOLECULAR BIOLOGY AND DIAGNOSTICS Mark Laurence A. Manzano, RMT TOPICS OF THE DAY 1 Definition of terms 2 Application and History 3 Introduction to DNA 4 Introduction to PCR  WHAT IS MOLECULAR BIOLOGY?  molecular biology studies macromolecules and the macromolecular mechanisms found in living things MOLECULAR BIOLOGY Study the organisms’ structure and physiology in molecular level (even smaller than cells). DIAGNOSTIC TESTING A procedure of examining a person’s physical and health areas to find their strength and weaknesses in order to assess their disease. MOLECULAR DIAGNOSTICS Molecular Diagnostics of the laboratory is considered a subset of the In vitro Diagnostic Testing because its analytes like protein and DNA are used in order to detect diseases that occurred during genetic mutation or alterations happened.  MOLECULAR DIAGNOSTICS The main goal of molecular Molecular Diagnostic is progressive and transformative reaching the point of diagnostics is to be examining of analyzing genetic materials incorporated to both and proteins responsible for disorders therapeutic management and present in the society. personalized medicine. MOLECULAR DIAGNOSTICS PERSONALIZED MEDICINE (PM) refers to addressing specific disorder unique to a certain individual by providing a personalized treatment that is specific to that disorder. There can also be uses of specific proteins or nucleic acids that can act as specific biomarkers to the disease. Proponent of this project is Barack Obama (44ᵗʰ president of USA).  PERSONALIZED MEDICINE 1. RISK ASSSESSMENT 2. SCREENING  PERSONALIZED MEDICINE 3. DIAGNOSIS 4. STAGING AND PROGNOSIS  PERSONALIZED MEDICINE 5. THERAPY SELECTION 6. MONITORING  Wild Type—normal DNA, RNA and base sequences in genetic MOLECULAR material. DIAGNOSTICS Gene Variant—Alteration in genetic sequence. IN CONTINUATION TO MOLECULAR DIAGNOSTICS, THE FOLLOWING TERMS SHOULD BE REMEMBERED: Polymorphism— Gene Variant that is normal, but the genetic code is uncommon.  In continuation to molecular diagnostics, the following MOLECULAR terms should be remembered: DIAGNOSTICS Mutation—a change that occurs in our DNA sequence, due to mistakes when the DNA is copied. 1. Hereditary – pass from parental 01 generation to offspring (congenital). 2. De novo – first time encountered mutation in a certain family due to 02 variants in the germ cell of either parents. 3. Somatic – The mutation that 03 happens after conception  2. HISTORY  S. pneumoniae S cells/ strain smooth colony (encapsulated and virulent) R cells/ strain rough colony ( non-encapsulated and avirulent) FREDERICK GRIFFITH In 1928, in an attempt to develop a vaccine against pneumonia, he discovered that bacteria could change from being avirulent to virulent  S. pneumoniae S cells/ strain smooth colony (encapsulated and virulent) R cells/ strain rough colony ( non-encapsulated and avirulent)  S. pneumoniae S cells/ strain smooth colony (encapsulated and virulent) R cells/ strain rough colony ( non-encapsulated and avirulent) Griffith called the protein "transforming principle". Today, what he observed was DNA of the S cells survived the heating process and taken up by the R cells. The R cells formed now a protective capsule In 1944, Oswald Avery, Colin Macleod, and Maclyn McCarty, in their study, they showed that the transforming principle Griffith discovered was DNA In 1952, DNA was confirmed further through the study of Martha Chase and Alfred Hershey. They used bacteriophage to determine if the genetic material they inject in E. coli is DNA.  In 1952, DNA was Sulfur-35 Radioactive label confirmed further through the study of Martha Chase and Alfred Hershey. They used bacteriophage to determine if the Phosphorus-32 Radioactive genetic material label they inject in E. coli is DNA. MOLECULAR 1949 01 BIOLOGY AND Characterization of sickle cell anemia as a molecular disease; Start coinage DIAGNOSTICS of the Molecular Disease; mutation to beta globulin chain HISTORY 02 1953 Discovery of the DNA double helix; Anti-parallelism due to the three components: sugar (hydrophilic)- phosphate backbone and nitrogenous bases (hydrophobic) MOLECULAR 1958 BIOLOGY AND ⚬ Isolation of DNA polymerases; replication of DIAGNOSTICS DNA inside the nucleus of the cell have 2 class: CLASS 1, CLASS 2. 1960 HISTORY ⚬ First hybridization techniques 1969 In situ hybridization 1970 DISCOVERY OF RESTRICTION ENZYMES AND REVERSE TRANSCRIPTASE MOLECULAR 1975 BIOLOGY AND ⚬ Southern Blotting DIAGNOSTICS 1977 ⚬ DNA Sequencing; Complementary Bases HISTORY 1983 ⚬ First synthesis of oligonucleotides ⚬ Can be used as primer or probe (10-20 base pair) 1985 ⚬ RFLP (Restriction Fragment Length Polymorphism; PCR (Polymerase Chain Reaction) ⚬ Traditional PCR: Qualitative PCR which only detects the presence of absence of analyte. ⚬ Kary B. Mullis: Discovered PCR and became Nobel Prize winner in Chemistry in 1993; Amplicon is product of PCR. ⚬ Isolated polymerase enzyme came from the E. coli. 1986 1988 Development of FISH Discovery if the thermostable THERMOSTABLE DNA polymerase (Fluorescence in Situ Hybridization) DISCOVERY: USED FOR HEAT RESISTANT DNA POLYMERASE; CAN CONTROL TEMPERATURE CHANGES ⚬ Denaturation: 90 C ⚬ Annealing: 50 C ⚬ Elongation: 70 C 1992 Conception of the real-time PCR DIFFERENT FROM THE TRADITIONAL PCR OF KARY B. MULLIS MOLECULAR 1993 BIOLOGY AND ⚬ Discovery of structure-specific DIAGNOSTICS endonucleases for cleavage assays ⚬ Nucleases: destruction of phosphodiester Bonds HISTORY 1996 ⚬ First application of DNA microarrays (DNA chip; TCCH)  HISTORY 2001 2002 First draft versions of the human genome Launch of the HapMap project (Haplotide sequence Application of protein profiling in Map): allows to find gene/genetic variation human diseases that affects health of people. 2003 2005 Final Draft of the Human Genome Project Introduction of high-throughput next- was finished. generation sequencing technology (single sample used for multiple gene probing analysis 2008 Launch of the 1000 Genomes Project (catalogue for human genomes) ⚬ Introduction of the CRISPR system for gene 2013 editing  HISTORY 2014 2015 ⚬ ANNOUNCEMENT OF THE SEQUENCING ⚬ Launch of the Precision Medicine ⚬ Barack Obama: Proponent. He based it on the idea OF THE HUMAN GENOME of blood transfusion where blood should be specific ⚬ ILLUMINA COMPANY CAN PRODUCE 600 to a certain person so medicine should be that way MILLION SEQUENCES PER DAY as well. ⚬ Genetic disorders came to light and is now being countered.  3. DEFINITION OF TERMS MOLECULAR ANALYSIS OF DNA & RNA; NUCLEIC ACID TESTING (NAT). DIAGNOSTICS EXTRACTION Isolation of DNA/RNA from other cellular components; lyse the cell to extract DNA/RNA GOLDEN STANDARD FOR EXTRACTION: 1. DNA: PHENOL CHLOROFORM ISOAMYL ALCOHOL 2. RNA: ACID PHENOL/LITHIUM CHLORIDE SALT SOL’N RNASE Enzyme that degrades RNA. Ubiquitous in environment. DNASE TARGET Enzyme that degrades DNA. Specific section of DNA under investigation; Chronic Myelogenous Leukemia (CML) (chromosome 9 or 22) DEOXYRIBONUCLEIC ACID Contains all the genetic 0 information on both prokaryotic and eukaryotic cells 1 The genetic information 02 is transferred from parent to daughter cells by DNA replication WALTHER FLEMMING, EDUARD STRASBURGER, EDOUARD VAN BENEDEN The first accurate counting of chromosomes are made. Cell division is observed. Terms chromatin, mitosis, cytoplasm, nucleoplasm, prophase and metaphase are coined.  DNA REPLICATION DNA Replication is semi-conservative. That means that when it makes a copy, one half of the old strand is always kept in the new strand. This helps reduce the number of copy errors. Occurs during interphase specifically S phase of the cell cycle  Central Dogma describes the transfer of genetic information within a cell. CENTRAL The process of DNA to RNA is termed transcription. DNA is used as a template for ribonucleic acid (RNA) synthesis. One strand of DNA is copied into messenger RNA DOGMA OF (mRNA) by RNA polymerase II. MOLECULAR The process of RNA to protein is termed translation. A molecule of mRNA is read BIOLOGY by ribosomal machinery in the cytoplasm resulting in the production of protein that perform cellular functions  MOLECULAR BIOLOGY The field overlaps with Molecular biology is the other areas of biology and study of biology at a chemistry, genetics, molecular level microbiology and virology Understanding the Applying in diagnosis the interactions between DNA, process of replication, RNA and protein synthesis transcription and as well as learning how translation of the genetic these interactions are material regulated DIAGNOSTIC MOLECULAR BIOLOGY OR MOLECULAR BIOTECHNOLOGY 01 The completion of the human genome project (1990-2003) has opened a myriad of opportunities to create new medicines and treatments 02 Integration of concepts in molecular biology with clinical laboratory techniques JAMES WATSON & FRANCIS CRICK (1953) They described the basic structure of DNA. Double helix (spiral) shaped with its sugar phosphate backbone on the outside and its bases on the inside; the two strand of helix run in opposite direction and are anti-parallel to each other. stabilized by hydrogen bonds between the bases (glue)  Double helix can be denatured to single- DNA stranded DNA through exposure to heat (94-98C) or chemicals, and then renatured through cooling (54-55C) or removal of DENATURATION chemical denaturants to allow the DNA strands to renature or anneal POLYMERASE CHAIN REACTION Laboratory technique used to make millions of copies of a particular region of DNA. The type of gene the researcher is interested in may be a genetic marker used by forensic scientists to match crime scene DNA with suspects. DNA amplified by PCR may be sent for sequencing, visualized by gel electrophoresis, or cloned into a plasmid for further experiments.  Reverse Transcription (RT) rRT-PCR or use an enzyme which converts RNA to DNA qRT-PCR Polymerase Chain Reaction (PCR) common technique for repeatedly amplifying/copying a specific segment of DNA to create enough copies for a signal to be detected Real time or quantitative (rt or q) the amount of light produced by the DNA copies will be counted TAQ POLYMERASE In 1992, Thomas Brock discovered Thermus aquaticus bacteria found in Yellow Stone National Park Its DNA polymerase is very heat-stable and ideal for PCR As we'll see, high temperature is used repeatedly in PCR to denature the template DNA, or separate its strands COVID-19 REAL TIME PCR KIT PRINCIPLE OF TEST: Detect the presence of 2 genes in the virus: ORF1ab and N gene. The signals can be amplified and detected based on the designed Taqman probes of the target genes during the amplification process