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

Microarrays

In situ Hybridization

Fluorescence in situ Hybridization

Immunohistochemistry

Flow Cytometry