Techniques in Molecular Biology and Biotechnology Sept 1, 2021 PDF

Techniques in Cell Biology, Molecular Biology, and Biotechnology Dr. Orlex B.Yllano College of Medicine Adventist University of the Philippines Biology in Different Scales (Castiglione et al., 2014) Cell Culture  Brightfield (unstained specime...

Techniques in Cell Biology, Molecular Biology, and Biotechnology Dr. Orlex B.Yllano College of Medicine Adventist University of the Philippines Biology in Different Scales (Castiglione et al., 2014) Cell Culture  Brightfield (unstained specimen) 50 µm Brightfield (stained specimen) Phase-contrast Microscopy Confocal Fluorescence Microscopy Fluorescence Fluorescence Resonance Energy Transfer (FRET) TEM Freeze-Fracture Replication and Freeze Etching SEM Atomic Force Microscopy The Use of Radioisotopes  Fractionation of Proteins and Nucleic Acids 1. Gel Electrophoresis 2. Ultracentrifugation Cell Breakage & Fractionation Density-Gradient Equilibrium Centrifugation Protein Separation Chromatography Gel filtration chromatography Affinity Chromatography X-ray Diffraction Analysis EM and X-Ray Crystallography Mass Spectrometry (Raad et al., 2018) Electrophoresis  Polyacrylamide Gel Electrophoresis (PAGE) (https://steemit.com; SA; Nuhair et al., 2019)  Separation of DNA Restriction Fragments by Gel Electrophoresis Two-Dimensional Gel Electrophoresis  Polymerase Chain Reaction selected DNA segment can be cloned or copy or amplified in a tube extremely sensitive (detect single DNA molecule in a sample) needs prior knowledge of the gene (Why?) easy and fast to do  PCR for Forensic  Components of PCR Mix Taq polymerase (DNA polymerase) primers (oligonucleotides) – one complementary to each strand of the DNA – lying on the opposite sides of the region to be amplified Magnesium salt selected DNA segment dNTP’s buffer  Stages 1. Denaturation - 94 0C 2. Annealing - 34 0C 3. Extension/Synthesis - 72 0C Then, the process is repeated which depends on the number of amplified copies needed. *~20-30 cycles Ex. of PCR Profiles and Amplification Reaction Conditions The optimized PCR reaction mixture contained 16.87μL sterile H2O, 2.5 μL 10X buffer with Mg2+, 2 μL 10mM dNTP, 1.25 μL of 10 μM primer (AM3 in the first set of samples and AM7 in the second set of samples), 0.13 μL of 5U/ μL Taq polymerase and 1 μL of 40 ng/μL of genomic DNA. The PCR profile had an initial denaturation at 94 0C for 2 min for one cycle, denaturation at 94 0C for 30 sec, annealing at 34 0C for 30 sec, extension at 72 0C for 30 sec for 45 cycles and final extension at 72 0C for 10 min for one cycle. The PCR run had a total of 47 cycles, which lasted for about 4-5h.  How? 1. Isolation of DNA from cells/tissues 2. Heated to separate its complementary strand 3. Two DNA strands are then annealed with the primers 4. Extension or synthesis  Applications amplifying segment of DNA cloning diagnosis of diseases (medicine and agriculture) forensic medicine PCR Animation  Restriction Enzymes Restriction endonucleases DNA cutting enzymes obtained from bacteria (function?) recognizes a specific sequence of 4-8 (usually 6) nucleotides ==>restriction site where it cleaves the DNa ~400  TYPES 1. Rare cutter 2. Frequent cutter depends on the occurrence of their recognition sites 3. Blunt-end cutter 4. Fragments with single stranded ends  Restriction Fragments DNA fragments of various sizes (restriction fragments) result from the digestion with a restriction enzyme. Restriction enzyme = cleave a given segment of DNA into fragments and diff. sizes diagnostic purposes  Restriction Map Is a linear sequence of restriction sites at defined intervals along the DNA essential in medical genetics breeding (e.g. marker aided selection) phylogenic researches Note: kindly see the accompanied photocopy DNA Sequencing Maps Cytogenetic Map  Genetic Maps Are representations of disease traits, physiological traits, or random genes assigned to a particular chromosomes and mapped relative to one another. - Markers are used to det. the distances of genes in the chromosomes - Centimorgans (cM) = used in measuring the genetic map; 1 million base pairs  Physical Maps Are not representations but overlapping collections of DNA fragments. DNA is snipped into fragments by the action of restriction enzymes, then cloned and stored in variety of forms such as plasmid bacteria or YAC. These tiny fragments (kb) may then be analyzed by various means to discover the base-by-base sequence of DNA. Much detailed than the genetic map. Libraries Genomic Library cDNA Library DNA Cloning Nucleic Acid Hybridization Knockout Mice  DNA Transfer into Eukaryotic Cells and Mammalian Embryos Growth Hormone Gene Chromosome Microdissection Spectral Karyotyping Monoclonal Antibodies DNA Mircroarray Technology  RNA Interference  Genome Editing Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)  https://doi.org/10.1080/13102818.2017.1406823 Hallmarks of CRISPR Gene Therapy (Uddin et al., 2020)  CRISPR Video https://www.youtube.com/watch?v=UKbrwPL3wXE&t=13s