Knowt
Lab Techniques
Enzyme-Linked Immunosorbent Assay (ELISA)
Purpose: Used to identify the concentration/presence of antigens (proteins or cytokines) or specific antibodies (immunoglobulins)
Can be used to screen patients for viral infections (sample taken from patient tumor biopsy or serum)
How it works:
uses primary antibodies that are specific to a molecule of interest and secondary antibodies (with detection enzymes) that are specific to the primary antibodies.
presence of molecule of interest will be measured via spectrophotometry or by a color change
Indirect ELISA
Molecule of interest in this case is an antigen.
antigen is coated within the wells of a plate
primary antibody is then added and washed to remove any unbound antibodies
a second antibody is added that contains a detection enzyme that causes a color change when reacting with an oxidizing agent
detection of color change can be done using photospectrometry and concentration of the molecule can also be determined
Sandwich Elisa
primary antibody is coated on the wells of the plate instead of the antigen (like in indirect)
molecule of interest is then added and washed to remove any unbound molecules
secondary antibody with detection enzyme is added, which changes color in presence of oxidizing agent
photospectrometry could be used to detect presence of a color change
Radioimmunoassay
Purpose: Used to determine the concentration of a protein of interest in a given sample
Mostly used to determine amounts of hormones or drugs are presents in a patients’ sera
How it works:
wells of plate are coated with the known amount of primary antibody that is specific to the protein of interest
a known amount radiolabeled protein, containing a tyrosine residue labeled with 125I is added to the wells and binds to the primary antibody
protein of interest in an unknown amount is then added and it completes for the active site on the primary antibody, displacing the radiolabeled protein
Gamma emission is taken before and after the protein of interest is added to measure how much radioactivity is measured
difference in concentrations provides the concentration of protein of interest
Gel Electrophoresis
Purpose: Separation of proteins, DNA, or RNA based on size and/or charge
Process:
Macromolecules of interest are placed in the lanes of a gel
gel is made of either acrylamide or agarose (for larger molecules of DNA)
At the bottom of the gel is a positively charged anode and at the top is the negatively charged cathode
An electrical charge is ran across the gel and negatively charged molecules will travel towards the anode
Due to the pores present within the gel, larger molecules will move slower than smaller molecules- causing the sample to be separated by size
Blue Coomassie dye can be added to stain the molecules so that they can be visually observed based on their size and how far they traveled
There are several applications of gel electrophoresis:
Native-PAGE
a polyacrylamide gel electrophoresis method for proteins that occurs under non-denaturing conditions
separates proteins by size while retaining their structure
SDS-PAGE
A polyacrylamide gel electrophoresis method for proteins that occurs under denaturing conditions to separate proteins by mass
negatively-charged sodium dodecyl sulfate (SDS) is added to denature the proteins
1 SDS is added for every 2 amino acids, giving all proteins the same charge-to-mass ratio
this allows them to separately sole on mass
smallest proteins are found towards the bottom of the gel
SDS only interrupts non-covalent bonds so disulfide bridges present within a protein will not be broken
useful for analyzing proteins with multiple subunits
Reducing SDS-Page
Similar to SDS-Page but uses a reducing agent to denature the proteins like beta-mercaptoethanol
reduces disulfide bridges and results in a completely denatured protein
Isoelectric Focusing
Separates proteins on the basis of their relative contents of acidic and basic residues
gel contains a pH gradient, low pH on one side and a high pH on another
proteins will travel toward the anode until they found an area that matches their isoelectric point
when a protein is at its pI, there is a net charge of zero on the protein so it would not move
Blotting
Overall premise: Used to isolate or detect a particular nucleic acid fragment or protein (called probing) once electrophoresis is ran
Typically uses a PVDR membrane to hold DNA/RNA/protein fragments and detection is done via radiolabeled probes (either radiolabeled antibodies or radiolabeled nucleic probes)
USE MNEUMONIC SNOW DROP TO REMEMBER BLOTTING TYPES AND WHAT THEY MEASURE
Southern Blotting: DNA
Northern Blotting: RNA
Western Blotting: Protein
Eastern Blotting: post-translational modifications of peptides
Recombinant DNA
Recombinant DNA: novel combination of DNA from different organisms, used to create recombinant RNA
Recombinant proteins: proteins that are obtained by transcribing and translating recombinant DNA
Very common procedures that are used a therapeutic agents in medicine
ex: Human insulin
gene for human insulin can be placed in a bacterial plasmid
the bacteria with the plasmid then produces human insulin
Advances in this technology is due to restriction endonucleases
restriction endonucleases recognize specific sequences of DNA and cut the molecule into two pieces
this allows the manipulation of genes to create recombinant DNA
Plasmids can uptake new chromosomes into their molecules and be absorbed by bacteria (bacterial transformation)
Artificial chromosomes can be used to carry large inserts
Plasmids that carry eukaryotic DNA, carry complementary strands of the needed eukaryotic DNA (since bacteria can’t splice)
cDNA is created from reverse transcriptase and carry the complete coding sequences for genes but lacks introns (making the sequence smaller than the genomic sequence of the gene) and allows for implementation into a bacterial plasmid
Eukaryotic plasmids also exist
they require the same components as bacterial plasmids but they use different selection agents and promoters in the expression of plasmids, as well as a poly-adenylation signal downstream to terminate transcription
introduced to mammalian host cells via transfection through chemicals, lasers, electroporation, or through the shooting of a cell nucleus using a gene gun
Polymerase Chain Reaction (PCR)
Purpose: to amplify a small quantity of DNA by several orders of magnitude
The amplification of DNA can be used for screening for hereditary and infectious diseases, cloning genes, and fingerprinting DNA
It is also very quick and inexpensive
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Used to detect the relative expression of specific gene products
DOES NOT MEASURE ACTUAL EXPRESSION OF ABUNDANCE OF PROTEINS
does measure the relative amount of target mRNAs
How its performed:
all mRNAs from a cell population are first isolated and then converted into complementary DNA using the enzyme reverse transcriptase
cDNAs is then subjected to PCR, using primers for a certain gene of interest
if the gene was actively transcribed at the time of harvest, its mRNA will have yielded cDNA, which will be amplified by the PCR reaction and visualized by the gel
Quantitative Polymerase Chain Reaction (qPCR)
PCR is both detected and quantified as either an absolute number of copies or as a relative amount normalized to a control
amplified DNA is detected in ‘real time’ as reaction progresses
detection can be either through a use of a dye that is fluorescent and binds to NDA or by a fluorescent oligonucleotide probe that hybridizes to the sequences of interest
Can be performed on DNA or cDNA and will given information on the presence and abundance of a particular DNA sequence in samples or on gene expression
DNA Sequencing
Purpose: Used to determine the sequence of nucleotides in a strand of DNA
provides the basis for investigating the genetics of health and disease
Most widely used DNA sequencing method is the Sanger technique
uses modified nucleotides known as ddNTPs (dideoxynucleotides) which allows termination of replication to be controlled
ddNTPs are missing the OH group on the 3’ carbon so they are unable to create a new 5→3’ phosphodiester bond
Process goes as follows:
DNA strand of interest is denatured using NaOH solution to create ssDNA strand that can be used for replication
ssDNA of interest is then added to a solution containing:
radiolableled DNA primer that is complementary to the gene of interest
DNA polymerase
dNTPs (dATP, dTTP, dCTP, dGTP) (depending which tube is holding which base)
small quantity of a single ddNTP
This step is done for each of the four nucleotides in separate solutions
Each solution is then place in their on lane on a gen and ran under gel electrophoresis
Gen is then transferred to a polymer sheet and autoradiography is used to identify the strands in a gel
The gen can be read from bottom-to-top to determine the nucleotide sequence
DNA Fingerprinting
Allows for scientists (and police departments) to detect sequence variations that make each individual’s DNA unique
Can be used to scan for a murder suspect (with DNA found at crime scene) or when screening for disease-causing genes or paternity testing
DNA exploits stretches of repetitive and high variable DNA called polymorphisms
polymorphisms are variable with respect to their sequence, length, multiplicity, and location within the genome
Two methods of finger printing include restriction fragment length polymorphism (RFLP) analysis and short tandem repeat (STR) analysis
Restriction length polymorphism analysis (RFLP)
uses restriction endonucleases to cut 10-100 base pair stretches of the polymorphic DNA (called minisatellites) into small fragments
because of the size variation inherent in this DNA, the resulting DNA fragments (referred to as RFLPs) also vary in size and are unique to the individual
The FRLPs are then separated using gel electrophoresis and then transferred onto a radiolabeled DNA oligonucleotide membrane and then southern blotting is used to analyze each sample
the membrane allows for the visualization of the RFLP sequences
Short tandem repeat analysis (STR)
Uses PCR to amplify 5-10 base pair stretches of highly polymorphic repetitive DNA located within intron regions of a genome
STRs vary with respect to the sequence and the number of repeats found at each locus
STRs are amplified using PCR, isolated by electrophoresis and then analyzed using southern blotting