MIC 205A III
Scientific Nomenclature
Carlos Linnaeus, system gives every organism 2 names which include the genus and the species
CRISPR
Cas9 (an endonuclease used for gene editing Uses single-stranded RNA (gRNA) to recognize specific DNA sequence Makes double-stranded cut at that DNA sequence DNA at that site can be altered by manipulating DNA repair mechanisms Disable genes (imprecise repairs) Edit genes (homology-directed repair)
culture characteristics
Colony morphology can give initial clues to the identity of an organism Includes: Colony size Colony colour Colony odour Differential media aids in identification
dideoxy chain termination
DNA sequencing technique that allows you to determine the sequence of bases in a piece of DNA
Pyrosequencing
DNA sequencing technology that is based on the generation and detection of a pyrophosphate group liberated from a nucleotide triphosphate
Recombinant DNA
DNA that has been formed artificially by combining constituents from different organisms.
Genotypic characteristics
Differences in DNA sequences can be used to determine the point in time at which two organisms diverged from a common ancestor
Taxonomic Hierarchy
Domain, Kingdom, Phylum, Class, Order, Family, Genus, Species
Genetically Engineered Eukaryotes
Engineered yeast for research and commercial products Transgenic plants have been engineered to resist pests and herbicides, have improved nutritional value, and function as edible vaccines.
Techniques of biotechnology
Enzymes: Restriction enzymes, ligases, DNA polymerases, reverse transcriptases, etc Vectors: used in cloning Transformation DNA/Protein gel electrophoresis Polymerase chain reactions RNAi CRISPR/Cas9
food production
GMO crops/microorganisms
Application of Biotechnology
Gene Editing Genetic Engineering Food production Diagnostic tests Gene therapies Precision/personized Medicine: Vaccines/Therapeutic proteins:
Culture
Grown in laboratory media
genetic engineering application
Health and agriculture, study expressions of particular genes, select traits that might benefit humans, study development of certain disease
Prokaryotic species
-Cells that share physical characteristics and have at least 70% DNA similarity -At least 97% identical 16S rRNA sequence similarity
Examples of Protein Production
-Human insulin expressed from bacteria -Proteins needed for vaccine production -Chymosin (rennin) for cheese production -Synthetic bacteria may express valuable products in future
cDNA libraries
-contain smaller fragments of DNA, and only include exons of genes expressed by the sample tissue -can be used to make recombinant proteins or for gene therapy
Number of archaeal phyla
26
Type of ribosome
70s for archaea and bacteria 80s for eukaryotes
Number of bacterial phyla
92
Biotechnology
A form of technology that uses living organisms, usually genes, to modify products, to make or modify plants and animals, or to develop other microorganisms for specific purposes.
whole genome sequencing
A method of genome sequencing that selects clones at random from a genomic library and after sequencing them, assembles the genome sequence by using software analysis
Sanger sequencing
A procedure in which chemical termination of daughter strands help in determining the DNA sequence.
Gram staining
A process by which components of bacterial cell walls are bound to Gram's stain. Depending on the amount of peptidoglycan in their cell walls, bacteria stain differently and are classified as Gram-negative or Gram-positive.
Cloning
A process in which a cell, cell product, or organism is copied from an original source.
isolate
A sample obtained from an infected individual, a specific site, etc
strain
A sub-population of a species that shares some properties absent in the rest of the species
variant
A sub-population that contains a number of nucleotide changes, may/may not have any change in properties
polymerase chain reaction (PCR)
A technique for amplifying DNA in vitro by incubating with special primers, DNA polymerase molecules, and nucleotides.
RNA interference (RNAi)
A technique used to silence the expression of selected genes. RNAi uses synthetic double-stranded RNA molecules that match the sequence of a particular gene to trigger the breakdown of the gene's messenger RNA.
genetic engineering
A technology that includes the process of manipulating or altering the genetic material of a cell resulting in desirable functions or outcomes that would not occur naturally.
Roche 454 Pyrosequencing
A type of "next generation sequencing": Questions of microfluidics, moving fluids around through small columns • Attach fragmented strands of DNA to little beads (certain fragment per bead, use primers to attach) and put in little holes and run fluids through them, producing lights that can sequence DNA • Similar to in vitro process of oligonucleotide synthesis - adds dNTPs one at a time, light will be produced when added - cheap, short, and reads fair amount of bases - read length = 500 bases, runtime (days per gigabase) = 2, cost per 1000 bases - $0.02
Intron presence
Absent in bacteria Present in eukaryotes Sometimes present in archaea
reverse transcriptase
An enzyme encoded by some certain viruses (retroviruses) that uses RNA as a template for DNA synthesis.
Ligase
An enzyme that connects two fragments of DNA to make a single fragment
clone
An organism that is genetically identical to the organism from which it was produced
genetically modified organisms
An organism whose genetic material has been altered through some genetic engineering technology or technique.
Cytoplasmic membrane lipids
Archaea - Hydrocarbons linked to glycerol by linkage Bacteria and Eukaryote- Fatty acids linked to glycerol by ester linkage
Nanopore sequencing
Involves passing a DNA sequence through a small nanopore and measuring the change in current (shape).
informal grouping of microbes
Lactic acid bacteria Anoxygenic phototrophs Endospore-formers Sulfate reducers
Identification
Matching characteristics of an "unknown" organism to lists of known organisms. Clinical lab identification, more practical Based on microscopic examination, culture characteristics, biochemical tests, nucleic acid analysis, or symptoms
PCR amplification
Melt: Highest temp, separates strands Anneal: Lowest temp, primers attach Extend: Medium temp, DNA replicates (Polymerase) Final Extension: Ten minutes at medium temp, ensure DNA has finished replicating
Ion semiconductor sequencing
Monitors H+ given off during addition of nucleotide to growing DNA molecule
What vectors contain
Multiple cloning sites Antibiotic resistance A secondary selectable marker
detecting specific nucleotide sequences
Nucleic acid probes and nucleic acid amplification tests can be used to identify prokaryotes grown in culture. In some cases, the method is sensitive enough to detect the organism directly in a specimen
Membrane bound nucleus
Only in Eukaryotes
Classification
Placing organisms in groups of related species. Lists of characteristics of known organisms, sequence similarities Bergey's Manual of Systematic of Archaea and Bacteria
peptoglycan cell wall
Present in bacteria and eukaryotes Absent in archaea
gel electrophoresis
Procedure used to separate and analyze DNA fragments by placing a mixture of DNA fragments at one end of a porous gel and applying an electrical voltage to the gel
genetic engineering
Process of making changes in the DNA code of living organisms
Five Kingdoms
Prokaryotae, protoctista, fungi, plantae, animalia
Next Generation Sequencing Strategies
Pyrosequencing Sequencing by synthesis Sequencing by ligation Ion semiconductor sequencing Nanopore sequencing
antibiotic resistance
Resistance evolving rapidly in many species of prokaryotes due to overuse of antibiotics, especially in agriculture.
Vector
Self-replicating DNA used to carry the desired gene to a new cell (plasmids, viruses, etc)
SOLiD Sequencing (NextGen)
Sequencing by ligation. A pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position. The oligonucleotides are annealed and ligated, the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR and the resulting beads (each containing single copies of the same DNA) are deposited on the glass slide for sequencing.
Fluorescence in situ hybridization (FISH)
Technique used to detect a given nucleotide sequence within intact cells on a microscope slide
colony blotting
Technique used to determine which colonies on an agar plate contain a given nucleotide sequence
sequence assembly
The process in which short nucleotide sequences of a long DNA molecule are arranged in the correct order to generate the complete sequence.
Taxonomy
The scientific study of how living things are classified
Sequencing rRNA genes
This requires amplifying and then sequencing rRNA genes, but it can be used to identify organisms that have not yet been grown in culture
acid fast stain
a differential stain used to identify bacteria that are not decolorized by acid-alcohol
gene editing
a highly precise type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using engineered nucleases
Metabolic capabilities
a set of biochemical tests can be used to identify a microorganism
Cassava Brown Streak Virus
a severe virus disease of cassava and prevalent in the eastern regions of Africa. The disease is characterized by distinct vein chlorosis and streak symptoms on leaves and stems and necrosis of storage roots. Requires the interaction between a viral-genome- linked protein (Vpg) and eIF4E for expression of viral genome Engineer eIF4E so that it no longer interacts with eIF4E, creating host immunity
protein profile
a technique for visualizing the proteins contained in a cell; obtained by the use of polyacrylamide gel electrophoresis
Ion Torrent
add each base one at a time and monitor release of proteins cross of PCR machine and micro-pH meter cheaper and no need to separate reaction products
Illumina sequencing
allows you to get billions of bases on something the size of a microscope slide
restriction enzymes
an enzyme produced chiefly by certain bacteria, having the property of cleaving DNA molecules at or near a specific sequence of bases. Name of the enzyme represents the bacterium the enzyme was isolated from Most restrictions sites range from 4 to 8 nucleotides Palindromes: the same on both strands when read in 5′ to 3′ direction
Bioformatics
application of mathematics and computer science to store, retrieve, and analyze biological data
dCas9
can be used along with a gRNA to silence a gene by blocking transcription
serological testing
can differentiate not only among microbial species, but also among strains within species
RNAseq
cellular RNA is isolated, converted to cDNA, and sequenced, providing information on the RNA transcripts present in a cell
DNA libraries
collection of clones containing all the DNA fragments from one source (tissue type, cell type, or single individual)
gene therapies
designed to treat or cure a variety of diseases by inserting a functional gene into an individual's cells to replace a defective version of the gene
Next generation sequencing
entire genomes sequenced using multiple parallel reactions to analyze short segments of DNA and compare the results to known sequences. Highly automated; fast, lower costs; analyze millions to billions of bases
Number of eukaryote kingdoms
five
microscopic morphology
fresh or stained microorganisms from specimen; shape, size, stain reaction, cell structures Quickly determine size, shape, and staining characteristics More useful to diagnose eukaryotic infections
Eukaryotic species
group of similar organisms that can sexually reproduce together
Diagnostics Tests
identify pathogens/diseases
Deoxy-chain termination sequencing
is based on the principle that during DNA synthesis, addition of a nucleotide triphosphate requires a free hydroxyl group on the 3′ carbon of the sugar of the last nucleotide of the growing DNA strand
PacBio sequencing
longest 10,000-70,000 nucleotides, 10-20% error, more expensive than Illuminia, more throughput than Sanger
Phenotypic characteristics
microscopic morphology, culture characteristics, metabolic capabilities, serology, fatty acid analysis
sequencing by synthesis
monitors identify which of 4 nucleotides is being added to complementary strand
viral species
population of viruses with similar characteristics that occupies a particular ecological niche
single cell genomics
refers to isolating individual cells, amplifying their genomes and sequencing these individual genomes one by one
Serotype
strain of bacteria that carries a set of similar antigens on its cell surface, often many in a bacterial species
Genomics
study of whole genomes, including genes and their functions
Phylogenetics
the analysis of evolutionary, or ancestral, relationships between taxa
Metagenomics
the study of genetic material recovered directly from environmental samples
transcriptiomics
the study of total metabolites produced
DNA Microarrays
tools that allow scientists to study many genes, and their expression, at once
Decoding Genomes
unlock the secrete of genes and genomes revolutionize our understanding of genetics, phylogeny Sequencing techniques change rapidly Human genome project
Sequencing by ligation
uses short fluorescently labeled oligomers that hybridize to complementary DNA template, flourescently labeled bases are used to ligate the sequence and are detected and the olignonucleotide is cleaved.
Other Applications of CRISPR Technology
• "Dead" or dCas9 does not cut DNA, but still uses RNA guide to bind to specific DNA sequence ◦ Tagged DNA modifying enzymes to modify specific nucleotides (editing) ◦ Tagged transcriptional factors promote gene expression ◦ May block RNA polymerase, shut off expression ◦ May deliver molecules to certain chromosome locations ‣ Fluorescent marker to locate a gene • CRISPR-based diagnostic tests: ◦ COVID-19 tests ◦ Cancer diagnosis
X-gal blue and white selection for recombinant DNA clones
• After transformation of bacteria with vectors carrying recombinant DNA, two layered selections allow for identification of recombinant bacterial clones
Vectors
• Independently replicate in an organism (replicon) • Can be plasmids, viruses, or artificial chromosomes • Carry new DNA to desired cell • Shuttle vectors can replicate in several different species • Ti (Tumor-inducing) plasmids of Agrobacterium tumefaciens insert DNA fragments to plant genome
Bacterial Defenses Against Invading DNA
• Restriction-Modification Systems • Evolved as a defense mechanism in bacteria • Restriction enzymes cut specific DNA sequences • Destroy bacteriophage DNA in bacterial cells • Modification enzyme protects cell's own DNA by adding methyl groups • Cannot digest (host) DNA with methylated cytosines
Hybridization with a probe
• The method used to detect specific nucleotide sequence in an unknown sample by using a gene probe • Gene probe is a short segments of DNA of a known sequence • A probe carries a radioactive label
Concerns for GMOs and other Biotechnologies
• Valid concerns: ◦ Allergens ◦ Spread of herbicide resistant gene to weeds ◦ Spread of BT toxin genes may harm non-targeted insects ◦ Misuse of personal DNA data (the Genetic Information Nondiscrimination Act) ◦ Creating a deadly pathogen (bioterrorism)? • Unfounded concerns: ◦ Causing cancer, liver damages ◦ Contain DNA ◦ Poke holes in guts • Antivaccine: ◦ contain tiny chips to control people ◦ Autisms