Methods in Microbiology Flashcards
Recombination can be used to ______ gene
delete
Deleting the gene helps you find out what the gene does
Wildtype gene
contains gene x ; a parental strain
the gene of interest
We can learn about what gene X does by comparing the ______ _______ stain to the _________ strain that lacks gene X
wild type
mutant
Ex. Testing to see how well they grow in a hot environment how sensitive they are to an antibiotic, or how well they survive without nutrients
Knockout gene
the DNA that is left once you take out gene X
Mutant stain
a strain with a mutation
Genotype vs phenotype
A genotype on an organism is the DNA sequence of its gene
A phenotype of an organism is its physical characteristics
Transposon mutagenesis
Can tell you what genes are required during a specific condition
- its essentially the gene that gets moved to a different DNA segment
PCR (polymerase chain reaction)
results in exponential amplification of the target sequence; uses DNA polymerase from a thermophile “heat loving” polymerase will remain stable at high temps
- amplify a gene for cloning
- identify a bacterial species
- detect a bacterium or virus in a low abundance sample
Is the basis for whole genome sequencing
Could you amplify DNA using DNA polymerase III from E. coli?
A. No, other components of the reaction, like the primers, get destroyed at 95°C.
B. Yes, but only if you amplify a gene from a non-thermophile.
C. Yes, but you’d have to keep adding the E. coli DNA polymerase after each round of heating.
D. No, E. coli polymerase only works on covalently
closed circular DNA molecules.
E. Yes, but it would only work for E. coli DNA.
C. Yes, but you’d have to keep adding the E. coli DNA polymerase after each round of heating.
DNA pol III is not thermostable and would be denatured at high temps
qPCR (quantitative PCR)
a molecular biology technique used to amplify and quantify specific DNA or RNA sequences
Ex: Nasopharyngeal swab for covid diagnosis
Gel electrophoresis
- Takes advantage of the inherent charge of nucleic acids
- separates DNA, RNA, or proteins based on SIZE by applying an electric current through a gel matrix, where smaller molecules migrate faster toward the positively charged electrode
Use a size standard, lane A, to figure out the sizes of other fragments
Visualize DNA using DNA dye and UV light
Nucleic hybridization
- relies on the ability of complementary strands of nucleic acid to hydrogen bond
- find a clone that contains a gene of interest (THIS IS CALLED A PROBE)
- identify related DNA sequences in other genomes
- See if a gene is expressed and how much its expressed
- Identify specific RNA or DNA sequence in intact cells
Southern hybridization (Southern blotting)
a technique used to identify specific DNA sequences in a complex mixture
- Separate DNA fragments by size (gel electrophoresis)
- Transferring them to a membrane
- Use a labeled probe to detect the target sequence via hybridization (two bands will show up instead of one where the target gene matches up with the probe)
Northern hybridization (Northern blotting)
Same thing as southern but with detecting RNA instead
Gene deletion can be used to determine the ______ of a gene by determining the mutant ___________
role
phenotype
Transposons are important tool for making ______ _________
mutant libraies
Carcinogenic
Causing mutation
PCR ,qPCR, RT-qPCR are used to _______ a specific target DNA sequence
amplify
Hybridization of nucleic acids
uses a probe that has a sequence that is complementary to the desired sequence - is conjugated to a signal
Restriction Enzymes
Restriction endonuclease - produced in diverse bacteria and archaea
- Cuts only double strand DNA targets, will not cut ssDNA, single strand RNA or double strand RNA
EcoRI (enzymes that cut)
Leaves sticky ends
EcoRV (enzymes that cut)
Leaves blunt ends
DNA _________ affects restriction enzyme activity
methylation
Ligation
using a phage DNA ligase, and some source of energy (ATP) to drive the reaction, heals the plasmid DNA sugar-phosphate backbone
Sticky vs blunt ends
sticky has overhang of bases which allows for complementary base pairing
blunt does not
Essential components of a plasmid for cloning and gene expression
- origin of replication
- MCS (region with many restriction enzyme sites)
- promoter
- selectable marker
Inducer
enhances or represses gene expression
MCS is placed in lacZ so that if insert goes into the plasmid the colonies will ______ have functional lacZ
not
Blue-white screening
Plate on good growth medium with ampicillin, IPTG, and X gal
blue colonies - LacZ has been made -lacZ gene has been reconstituted
white colonies - functional Lac Z is not made - a chunk of DNA has been inserted into the plasmid and disrupted lacZ
