Molecular Biology Lecture Notes PDF

26 12th lecture Molecular medicine - Promotes the understanding of normal body functioning and disease pathogenesis at the cellular and molecular level - Allows researchers and physicians to apply this knowledge to the disease diagnosis, treatment, prognosis and preven...

26 12th lecture Molecular medicine - Promotes the understanding of normal body functioning and disease pathogenesis at the cellular and molecular level - Allows researchers and physicians to apply this knowledge to the disease diagnosis, treatment, prognosis and prevention In vitro methods in diagnostics Omics - Genomics – DNA - Transcriptomics – mRNA - Proteomics – proteins - Metabolomics – metabolites (amino acids, organic acids, carbohydrates etc.) Genomics Polymerase chain reaction (PCR) - Is widely used to amplify DNA regions of known sequences - The basic method of molecular biology  27 PCR applications - Genetic research - Medicine – diagnostics - Microbiology and virology - Mycology and parasitology - Forensic science - Environmental science Gel electrophoresis - Agarose gel electrophoresis: o Agarose is complex sugar derivate from seaweed, gel is made of long chains of interlinked sugars, polymerised by cooling o Horizontally run o Resolution (separation) 50 – 30000 bp of DNA (different concentration of gel) - Polyacrylamide gel electrophoresis (PAG): o PAG is made by chemical crosslink of acrylamide and bisacrylamide o Vertically run o High resolution (separation) for shorter DNA fragments of 5-500 bp length Diagnostics of CF using PCR and gel electrophoresis - Cystic fibrosis is caused by allelic variants in a CFTR gene, the most common allelic variant worldwide is p.Phe508del, which occurs in approx. 75% of patients with CF, pPHe508del allelic variant is 3 nt deletion (in frame) which leads to missing Phe in position 508 Restriction Fragment Length Polymorphism (RFLP) - Used as direct method for analysis of allelic variants (may be applied in different ways) - Restriction enzymes are DNA-cutting enzymes found in bacteria, a restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides Restriction enzymes – recognition site Work flow in RFLP - Sample → Isolation of DNA → PCR → Incubation with enzyme → Electrophoresis → RFLP analysis  28 Diagnostics of LCHADD using RFLP and electrophoresis - Long chain 3-hydroxyacyl-Co-A dehydrogenase deficiency o Caused by allelic variants in HADHa gene encoding long-chain hydroxacyl-CoA dehydrogenase, enzyme catalalyzes steps in mitochondrial beta-oxidation of fatty acids o Clinical symptoms – early-onset, cardiomyopathy, hypoglycaemia, neuropathy, sudden death o The most common allelic variant is 1528G>C which results in Glu substitution with Gln in 510 position is directly responsible for the loss of dehydrogenase activity without changing the structure of enzyme complex Sanger (direct) sequencing - Named after Fred Sanger, who, with Walter Gilbert, received the Nobel Prize in 1980 for developing DNA sequencing method - Method to detect sequence of DNA fragment (up to 800-900 bp long)  29 Work flow in Sanger sequencing - Sample → Isolation of DNA → PCR → Sequencing reaction → Capillary Electrophoresis → Data analysis – reference genome Next Generation Sequencing (NGS) - Provides a much cheaper and higher-throughout alternative to sequencing DNA than traditional sanger sequencing - High-throughput sequencing of the human genome facilitates the discovery of genes and regulatory elements associated with disease - Targeted sequencing (gene panels) allows the identification of disease-causing allelic variants for diagnosis of pathological conditions - Provides a deeper coverage of genomic regions of interest Basic workflow for NGS  30 Limitations of NGS - Although much less costly in time and money in comparison to Sanger sequencing, it is still expensive for many labs - Inaccurate sequencing of repeated nucleotide regions on certain NGS platforms can lead to sequence errors – quality data for diagnostics - Data analysis can be time-consuming and requires special knowledge of bioinformatics to acquire accurate information from sequence data Transcriptomics PCR modifications - Reverse transcription PCR (RT-PCR): o Technique for mRNA detection and quantitation  31 - Quantitative PCR (qPCR): o Combines PCR amplification and detection into a single step o Fluorescent dyes are used to label PCR products during thermal cycling o Instrument measure the accumulation of fluorescent signal during exponential phase of the reaction – in real-time Real-time PCR Real-time qPCR Example – transcriptomic - Sample → isolation of mRNA → RT-PCR → Real-time qPCR → Data analysis → Differential gene expression Metabolomics and proteomics Why proteomics and metabolomics? - Protein coding genes in human genome – approx. 22000 – 250000 - Human genome – approx 1000000 proteins - Human metabolome – approx. 5000 metabolites Work flow in proteomics - Sample → Isolation of proteins → Separation – electrophoresis, liquid chromatography (LC) → Detection - mass spectrometry (MS) → Identification – database → Differential protein concentrations Work flow in transcriptomics - Sample → Extraction of metabolizes → Separation – electrophoresis, liquid chromatography (LC), gas chromatography (GC) → detection – mass spectrometry, nuclear magnetic resonance (NMR) → identification – database → differential metabolite concentrations  32 Case description - Patient 1y 9m old boy with unclear aetiology hypoglycaemia and hypocalcaemia hospitalizes in University Children’s Hospital Organic acids spectra analysed by GC/MS Confirmation of diagnosis – RFLP - Genotype: 1528G>C/1528G>C Precision (personalized) medicine - Refers to the tailoring of medical treatment to the individual characteristics of each patient. …the ability to classify individuals into subpopulations that differ in their susceptibility to a particular disease or their response to a specific treatment. Preventive or therapeutic interventions can then be concentrated on those who will not benefit, sparing expense and side effects for those who will not Treatment of CF - pPHE508del allelic variant: this deletion produces a partially unfolded CFTR protein that is retained in the endoplasmic reticulum and diverted to degradation by the proteasome (class II)  33 - Corrector molecules increment in the surface expression of pHE508del CFTR channel - G551D allelic variant: the CFTR protein reaches the cell surface, but the ability of the channel to open is impaired (class III) - Restoring chloride transport to Phe508del CFTR require correction (Lumacaftor) of cellular misprocessing to increase the amount of functional mutated CFTR - Potentiation (Ivacaftor) helps to further increase channel opening in most of class III (gating) allelic variants  34 DNA alteration specific treatment in cancer - Alteration of DNA sequence in somatic cells: o Amplification and overexpression in FGFR2; VEGF/VEGFR; EGFR; HER-2 etc. genes o Allelic variants in kRAS; BRAF, PI3K etc. genes Targeted therapy in gastric cancer - Targeted chancer agents: o Therapeutic monoclonal antibodies – target specific antigens found on the cell surface, such as transmembrane receptors or extracellular growth factors o Small molecules – penetrate the cell membrane to interact with targets inside a cell, are designed to interfere with the enzymatic activity of the target protein  35 Biotechnology - The use of living organisms or biological processes for the purpose of developing useful agricultural, industrial or medical products, especially by means of techniques such as genetic engineering, that involve the modification of genes - Medical biotechnology is also known as red biotechnology and deals with the development of new diagnostic and therapeutic procedures Biopharmaceuticals produced by biotechnology processes - Antibiotics, blood clotting factors, hormones, cytokines, growth factors, enzymes, vaccines - Current biotechnological processes essentially involve five different groups of organisms: o Bacteria (e.g. Escherichia coli) o Fungi (e.g. Saccaromyces cerevisiae) o Plants (e.g. tobacco plant) o Insects (e.g. Spodoptra frugiperda) o Mammalians (e.g. Chinese hamster ovary cells (CHO), baby hamster kidney cells (BHK) and transgenic animal cell lines Percentage of biopharmaceuticals produced in different expression systems Recombinant human insulin - The first licensed drug produced using recombinant DNA technology was human insulin, which was developed by “Gentech” and licensed as well as marketed by Eli Lilly in 1982 - Recombinant human insulin has been produced predominantly using E.coli and Saccharomyces cerevisae Human insulin - Synthesized as a single polypeptide known as preproinsulin in pancreatic beta cells - Preproinslin harbours a 24-residue signal peptide, which directs the nascent polypeptide to the ER - The signal peptide is cleaved as the polypeptide is translocated and forms proinsulin - In ER the proinsulin is folded in proper confirmation with the formation of 3 disulphide bonds  36 - Folded proinsulin is then transported to the trans-Golgi network, where it is converted into active insulin by cellular endopeptidases Biosynthesis of proinsulin Steps in recombinant insulin production - E.coli is a preferred microorganism for large-scale production of recombinant proteins - Various post-translational modifications do not occur in E. coli Recombinant DNA technology Recombinant insulin production Recombinant insulin - Humulin, Novolin, Velosulin - Recombinant human insulin is available in different concentrations under different forms of therapeutic action (insulin lispro, insulin aspart, insulin glargine – respectively, very fast, fast, long acting)