DNA Technology 2025 PDF

WE MAKE DOCTORS DNA technology Ana Gabriela Colima Fausto, PhD Learning objectives Explain and summarize relevant molecular techniques for the analysis of problems related to biological systems Name and describe techniques that can be used to confirm a clinical diagnosis and to perfo...

WE MAKE DOCTORS DNA technology Ana Gabriela Colima Fausto, PhD Learning objectives Explain and summarize relevant molecular techniques for the analysis of problems related to biological systems Name and describe techniques that can be used to confirm a clinical diagnosis and to perform mutational screening Name and describe the molecular techniques used for the diagnosis of chromosomal abnormalities. DNA Technology Clinical Diagnosis Association studies Forensic Medicine Pharmacology Agriculture Bionformatic Anthropology Evolution Population genetics Biotechnology Gene therapy Prenatal and Newborn Diagnosis  DNA Technology Vaccines: altering the pathogen's genes and mimicking surface proteins of harmful pathogens. Therapeutic hormones, such as insulin and human growth hormone. Genetically modified microbes are used in bioremediation. Insect resistance Transgenic foods Estimate cancer risk or to identify markers that may be associated with disease Paternity Test DNA Technology Obtaining nucleic acids DNA extraction and RNA extraction Different methods: Miller, CTAB-DTAB, phenol-cloroform Cells and tissues: blood The cells need to be lysed to release their components. The nature of the treatment will vary widely according to the celll type (Detergent) Removal of proteins Separate the DNA from other components Precipitate the DNA: Alcohol DNA Technology Electrophoresis A charge molecule is placed in an electric field, will migrate towards the electrode with the opposite charge, DNA, being negatively charged, will move towards the positive pole (anode). In a gel, which consists of a complex network of pores, the rate at which a nucleic acid molecule moves will be determined by its ability to penetrate through this network. DNA Technology Amplification of DNA PCR, 1993 by Kary Mullins PCR uses Taq polymerase for the exponential amplification of a DNA fragment from a template. The amplified fragment is defined by two short synthetic oligonucleotides that are complementary to the opposing DNA straand of the template that is being amplified. DNA Technology Sequencing Determine the sequence of bases of which is composed Sanger: dideoxy sequencing DNA sequencing machines The primer or the ddNTPs are labelled by incorporation of fluorescent dye Four ddNTPs are labeled with different dyes, the sequencing reaction is carried out in the same tube and then separated by gel electrophoresis in a capillar inside the automatic sequencers. The sequence will be derived by reading and assembling several overlapping sequences. DNA Technology DNA Technology Next generation sequencing (pyrosequencing) Large genomic deletions of exons or whole genes and rearrangements such as inversions and translocations Personnel expertise required to comprehensively analyse and interpret the subsequent data Expensive cost Multiplex ligation-dependent probe amplification One of the most accurate, time-efficient techniques to detect genomic deletions and insertions DNA methylation Techniques for gene expression Microarray: Measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome qRT-PCR: Quantifies the copies of an RNA transcript (mRNA) A substance marked with a fluorophore is added to this mixture in a thermal cycler that contains sensors for measuring the fluorescence of the fluorophore. Western Blot (Protein analysis) Western blot is often used in research to separate and identify proteins. In this technique a mixture of proteins is 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. The membrane is then incubated with labels antibodies specific to the protein of interest. Western blot USES: Measuring the amount of antigens or antibodies Confirmation of the presence of a protein in a sample Amount of protein in a sample Southwesternblot: target proteins binding to radiolabeled double-stranded DNA probes Uses: study of DNA-binding proteins (e.g., transcription factors, such as c-Fos, c-Jun) Chromosomal abnormalities FISH (fluorescence in situ hybridization) A labeled single-stranded DNA segment (probe) is exposed to denatured metaphase chromosome Useful for detection of small insertions and deletions Fluorescense microscope CGH (comparative genomic hybridization ) Scanning for deletions and duplications of chromosome material in cancer cells: cancer DNA is extracted from cells from a tumor. The DNA is then labeled with a substance that exhibits one color under a fluorescence microscope; DNA taken from normal control cells is labeled with a second color. USES: ✓ Direct visualization chromosomal anomalies at of FISH specific gene loci on a molecular level ✓ Philadelphia chromosome translocation t(9;22) AMBOSS GmbH.Laboratory medicine.https://amboss.com/. Accessed January 8th, 2025. CGH Preparation of the chip Thousands of genetic sequences of nucleic acid (DNA or RNA) probes are attached to a chip (e.g., glass, silicon). Both patient and control DNA is applied to this chip and hybridizes to the probes on the chip. The chip is mounted to a scanner that can detect complementary binding of probes and sample sequences The higher the expression of the gene in one sample, the more intense the fluorescence. Uses Helpful for detection of copy number variations (CNVs), single nucleotide polymorphisms (SNPs) that are used for genotyping, forensic science, cancer mutations, genetic linkage analysis, and genetic testing. Bibliography AMBOSS GmbH. Laboratory medicine. https://amboss.com/