Lecture 12 Flashcards
Omics (4):
Genomics – DNA
Transcriptomics – mRNA
Proteomics – proteins
Metabolomics – metabolites (amino acids, organic acids, carbohydrates etc.)
The polymerase chain reaction (PCR) is widely used to amplify:
DNA regions of KNOWN sequences.
The basic method of molecular biology.
PCR steps:
Denaturation, primer annealing and DNA synthesis
PCR applications:
Genetic research, Medicine - diagnostics, Microbiology and virology, Mycology and parasitology, Forensic science, Environmental science.
Agarose vs polyacrylamide gel electrophoresis?
Agarose is ran horizontally (useful in laboratories). It is easy to cast, easy to extract DNA fragments.
Polyacryalamide is vertically ran. High resolution for SHORTER DNA fragments!
Diagnostics of CF using PCR and electrophoresis example:
Cystic fibrosis (CF; MIM #219700) is caused by allelic variants in a CFTR gene. The most common allelic variant worldwide is p.Phe508del, which occurs in ~75% of patients.
p. Phe508del allelic variant is 3 nt deletion (in
frame) which leads to missing Phe in position 508.
Restriction Fragment Length Polymorphism (RFLP) is 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.
Sanger (direct) sequencing used for?
Method to detect sequence of DNA fragment (up to 800 – 900bp long)
Next Generation Sequencing (NGS):
Cheaper and higher-throughput alternative to sequencing DNA than Sanger.
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.
Limitations of NGS:
Inaccurate sequencing of repeated nucleotide regions on certain NGS platforms, can lead to sequence errors – quality of data for diagnostics!
Data analysis can be time-consuming and requires special knowledge of bioinformatics to acquire accurate information from sequence data.
Reverse transcription PCR (RT-PCR):
technique for mRNA detection and quantitation.
Quantitative PCR (qPCR):
Combines PCR amplification and detection into a single step.
Fluorescent dyes are used to label PCR products during thermal cycling
Workflow in RFLP:
Sample -> Isolation of DNA -> PCR -> Incubation with enzyme -> electrophoresis -> RFLP analysis
Workflow in Sanger sequencing:
Sample -> Isolation of DNA -> PCR -> Sequencing reaction -> Capillary electrophoresis -> Data analysis - reference genome
Why proteomics and metabolomics?
Protein coding genes in human genome – approx. 20 000 – 22 000.
Human proteome – approx. 1 000 000 proteins.
Human metabolome – approx. 5 000 metabolites.
Targeted cancer agents in precision medicine?
Therapeutic monoclonal antibodies -> target specific
transmembrane receptors.
Small molecules - penetrate the cell membrane to interact with targets inside a cell. Small molecules are designed to interfere with the enzymatic activity of the target protein.
What is 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.
Biopharmaceuticals produced by biotechnology processes examples?
Antibiotics Blood clotting factors Hormones Cytokines Growth factors Enzymes Vaccines
Current biotechnological processes essentially involve five different groups of organisms:
Bacteria Fungi Plants Insects Mammalians
Gel electrophoresis is a technique used to?
Separate DNA fragments according to their size.
DNA fragments are negatively charged, so they move towards the positive electrode.
Shorter molecules go faster in the cell!
Contrast between situations when DNA extraction and when RNA extraction should be used for diagnostic purposes?
DNA extraction: genetic diseases, structure of genome.
RNA extraction: gene expression, how active is a gene.
Why is recombinant insulin production two different bacterial cultures should be used?
Active insulin consists of two subunits which are made through specific post-translational modification in eukaryotes.
Since bacteria are unable to perform the same modification, two different cultures should be used and then product proteins combined.
How to make recombinant protein? Steps:
Euraryotic cell
mRNA extracted
Reverse transcription
cDNA acquired from mRNA
Bacterial cell
Plasmid
Extracted plasmid
Plasmid cut using restriction enzyme
Plasmid ligated with the target DNA
Recombinant plasmid ‘‘placed’’ in bacterium
Transformed bacteria are grown in a culture
Produced protein is collected and purified.
