Chapter 10 & 17 - An Overview of Research Uses and Laboratory Diagnostics of Microbiology

Genomics

Study of molecular organization of genomes, their information content, and gene products they encode for

What does genomics determine?

identification of species present and provides evolutionary insights

What does the genome tell you?

the sequence of amino acids; shows potential gene products

Transcriptomics

Study of the transcription of genes, presence of mRNA and quantity throughout time or location of mRNA

What does transcriptomics determine?

Determines how gene expression is altered in response to environmental changes; time sensitive

Proteomics

The study of the proteome, or the entire collection of proteins that an organism produces; Provides information about genome function not available from mRNA studies; studies 3D structure and function

What does proteomics determine?

Function of different cellular proteins, how they interact with one another and other molecules within the cell, and the ways in which they are regulated

Metabolomics

The study of all the metabolites, or the entire collection of macromolecules that an organism produces; provides information about the interactions between macromolecules; deals with things outside of the central dogma pathway

Which research method is the broadest, most difficult to use, and is very specific?

metabolonics

What are the types of genetic engineering?

Bioengineering and biotechnology

Bioengineering

direct, deliberate modification of an organism’s genome

What are the types of bioengineering?

Recombinant DNA technology and DNA cloning

Recombinant DNA technology

DNA molecules with segments originating from different organisms

DNA Cloning

use of enzymes and bacterial cells to modify and amplify DNA, segments originate in original organism

Biotechnology

use of an organism’s biochemical and metabolic pathways for industrial production

How are many enzymes in research produced or isolated?

via microbes

Restriction endonuclease

recognizes specific sequences of DNA and breaks phosphodiester bonds between adjacent nucleotides, (Exclusive to prokaryotes)

Restriction endonuclease are exclusive to:

prokaryotes

Polymerase

builds a Nucleic Acid Sequence

DNA Polymerase

makes a DNA copy from DNA template

RNA Polymerase

Makes RNA from DNA template

Ligase

necessary to rejoin phosphate-sugar bonds (sticky ends)

Reverse transcriptase

makes a DNA copy of RNA (cDNA); exclusive to viral microbes; Synthesizes double-stranded DNA from RNA template (mRNA) Used to construct complementary DNA (cDNA)

Reverse Transcriptases are exclusive to

viral microbes

What are the uses of restriction enzymes for research?

CRISPR, recombinant technologies, forensic science (RFLP analysis and DNA fingerprinting

Restriction Fragment Length Polymorphism (RFLP)

allows for DNA splicing at a palindromic site; cuts are made in distinct areas in genomes in very specific locations

Restriction Enzymes

Recognizes, binds, & cleaves specific sequences in DNA called palindromic sites; results in sticky ends or blunt ends in the DNA target; the DNA is in various size pieces called restriction fragments

Sticky end

provides overhang for the insertion of new genes

Blunt end

deactivates genes

What are the uses of polymerase?

research applications (Can isolate and amplify particular sequences of DNA from complex materials such as soil, water, and blood), diagnostic testing (only identifies the sequences of DNA you are looking for; very rapid, sensitive, and specific), and forensic sciences (fingerprinting technology)

Polymerase Chain Reaction (PCR)

a method to amplify DNA; rapidly increases the amount of DNA in a sample

What is the purpose of PCR?

it determines the presence of absence of specific DNA

What are the steps of PCR?

Denaturation, annealing, and extension

Denaturation

first step of a PCR in which a solution containing double-stranded DNA is heated to separate the DN into two individual strands

Annealing

the second step of a PCR in which the solution containing the DNA is cooled, and the two primers that are added anneal to their complementary sequence on the strands of the template duplex

Extension

the third step of a PCR in which DNA polymerase synthesizes new DNA stands (complementary to the template duplex strands) by extending primers in a 5’ to 3’ direction

What are the components necessary for PCR?

Template DNA, DNA polymerase (thermally stable), the four dNTPs (A,T,G,C), two primers (Must know general flanking sequence)

What is a dNTP?

an activated nucleotide to be attached to polymerase

Rt-PCR

a type of PCR done with cDNA that determines the quantitative amount of DNA in a sample; Used to measure and monitor microbial load in DNA viruses or for drug susceptibility testing

Drug Susceptibility Testing

Compare microbial (viral) load measurement after treatment to baseline measurement using a known Ct value

DNA Sequencing

determining the Nucleic Acid sequence of DNA

What is the most common method of DNA sequencing?

Sanger sequencing

Sanger Sequencing

determines if there has been a change in the amino acid sequence (qualitative);Test strands are denatured to serve as a template to synthesize complementary strands dNTPs are fluorescently labeled and the addition of each ddNTPs can be tracked via color; different colors tell which base pairs are present

What are the steps of Sanger Sequencing?

PCR with fluorescent chain-terminating dNTPs, size separation by capillary gel electrophoresis, laser excitation & detection by a sequencing machine

Next Generation DNA Sequencing (NGS)

cheaper, faster way of obtaining large amounts of sequence information, doesn’t require known primers; shears DNA into many pieces and attaching oligonucleotide adapters to their ends using ligase

What are the steps for NGS

extraction, library prep, sequencing, and analysis

Oligo

region of known DNA sequence that can function as a sequence that you want to amplifyqq

What is the purpose of ligase in recombinant DNA technology?

the intentional removal of genetic material from one organism and combining it with that of a different organism

What are the uses of ligase?

gene insertion

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

a natural system observed in EuBacterial prokaryotes that has been adopted for gene editing; a natural prokaryotic defense system; acts as an adaptive immune system; generates a memory that is inheritable

How does CRISPR work?

a bacteria under viral attack searches through spaces to ID matching viral DNA; blunt cuts are made to the viral DNA, inactivating it

What are the enzymes used by CRISPR?

restriction enzymes (splicing), helicase (unwinding), ligase (attatching), polymerase (copies)

What is different about CRISPR adaptive immunity vs Human adaptive immunity?

CRISPR adaptive immunity is inheritable, but human is not

CRISPR-Cas9 as a biotechnology

Link together the Restriction Enzyme with a gRNA and tells the DNA where to cut

Guide RNA (gRNA)

a genetic sequence that matches the piece of DNA to be modified by CRISPR

What are the two methods of repairing a cut in CRISPR?

Gene disruption and DNA insertion

Gene disruption

method of repair using CRISPR in which repair to the native sequence results in frameshift or mutations

DNA Insertion

method of repair using CRISPR in which a promoter, gene tag and single or multiple genes are inserted

Bioinformatics

Analysis of genome data using computers

What can you use bioinformatics for?

generate data on genome content (gene annotation), functional genomics, and evolutionary insight

Gene Annotation

process that locates genes in the genome map; Infer data on protein structure and function; Identifies each open reading frame in genome

Functional Genomics

maps the structure and arrangement of various genes; physical maps are used for determining the location of genes; provides information on metabolic pathways, transport mechanisms, and regulatory and signal transduction mechanism

Gene Families

genes that provide benefits and have been copied and aced on by natural; Orthologous or paralogous

What does gene duplication do?

increases # of genes in the genome; provides more opportunities for evolutionary changes; can result in gene families

Orthologous Genes

present in both species of interest because a common ancestor had it; Found in a single copy in the genome; homologous between species; can diverge only after speciation occurs

Paralogous genes

a gene gets duplicated in an organism, and one is modified because of environmental pressure; results from gene duplication; found in multiple copies in the genome; can diverge within the clade that carries them; often evolve new functions

Comparative Genomics

Set of analyses by which gene function and evolution can be inferred by studying similar nucleotide and amino acid sequences found among organisms; comparisons within
domains; comparisons of genomes of strains within species and among species

What are examples of comparative genomics in microbial groups?

Horizontal gene transfer (HGT), genomic islands (permanently integrated mobile genetic elements), pathogenicity islands (virulence proteins), phylogenic relationships between
microbes can be studied by synteny—order of genes on genome

Metagenomics

Study of microbial genomes based on DNA extracted directly from the sample environment, or community sample; allows one to learn more about the diversity and
metabolic potential of microbial communities; can determine the presence and level of classes of genes

Why is the extraction of RNA directly from the sample environment or community sample,
followed by sequencing and comparison to known sequences technically challenging?

due to the instability of RNA vs DNA molecules

How is a proteome often analyzed?

by 2D gel electrophoresis

What does analysis of a proteome also you to determine?

which proteins are present

Protein Modeling

determines the 3D structure of proteins and predicts their structure

Lipidomics

analysis of a cell’s lipid profile

Glycomics

analysis of a cell’s carbohydrate profile

Metabolomics

analysis of a cell’s small molecule metabolites from all macromolecule

What are omics and enzymes used for?

diagnostics and information gathering

What is the first step in disease identification and treatment?

identification of a microorganism

Specificity of a test

property of a test to only be positive a singular pathogen of interest and not react with any others

Sensitivity of a test

property of a test to even detect small amounts of the pathogen

Specificity of a test reduces false ______

positives

Sensitivity of a test reduces false _______

negatives

What are the approaches for laboratory identification and diagnosis of microbial infections?

Microscopy, Culture, Nucleic acid detection, Antibody detection, and Antigen Detection

Microscopy

Looking for identification via Phenotypic attributes (morphological-Structural); often uses staining techniques

What are the two different types of microscopes?

light microscopes and electron microscopes

CPE

Cytopathic effects (damage to host cells)

What is the importance of using for cytopathic effects of microbes on host cells?

for diagnostic purposes to determine the presence of a microbe

What doe CPE’s on inclusion bodies look like and indicate?

Subtle intracellular abnormalities; specific viruses

Hemagglutination

Adherence of red blood cells to other cells, including virally infected cells

Culture

Generally looking for identification via phenotypic attributes; Physiological/Biochemical; often uses staining techniques and selective media methods

Culture and plaque assays

Quantitative assay measuring number of microbes in a prepared virus stock; uses Microbial Load Testing and Drug Susceptibility Testing; gold standard for measuring the effects of antiviral drugs

Nucleic Acid Detection

Looking for identification via genotype presence; Nucleic Acid-Amplification Tests (NAATs

Antibody Detection

Presence of antibodies are an indirect measure of infection; patient serum contains antibodies; recent viral infection: IgM; re-infection with same virus: IgG

What are the methods of antibody detection?

Indirect immunofluorescent assays, Indirect ELISA, Western blot

Antigen Detection

uses Enzyme-linked immunosorbent assay (ELISA)