Nanosequencing

-based on the electrical characterization of individual nucleobases


A nanopore is simply a small hole, of the order of 1 nanometer in internal diameter. Certain porous transmembrane cellular proteins act as nanopores, and nanopores have also been made by etching a somewhat larger hole (several tens of nanometers) in a piece of silicon, and then gradually filling it in using ion-beam sculpting methods which results in a much smaller diameter hole: the nanopore. Graphene[3] is also being explored as a synthetic substrate for solid-state nanopores.

The theory behind nanopore sequencing is that when a nanopore is immersed in a conducting fluid and a potential (voltage) is applied across it, an electric current due to conduction of ions through the nanopore can be observed. The amount of current is very sensitive to the size and shape of the nanopore. If single nucleotides (bases), strands of DNA or other molecules pass through or near the nanopore, this can create a characteristic change in the magnitude of the current through the nanopore.


Alpha hemolysin (αHL), a nanopore from bacteria that causes lysis of red blood cells. bind an exonuclease onto the αHL pore. The enzyme would periodically cleave single bases, enabling the pore to identify successive bases. Coupling an exonuclease to the biological pore would slow the translocation of the DNA through the pore, and increase the accuracy of data acquisition.

homomeric αHL pore that has a ring of seven ardinines
located near the barrel of the pore.  To bring the diameter of
the conducting pathway closer to the size of the DNA a
cyclodextrin adapter is fitted within the pore.

Mycobacterium smegmatis porin A (MspA) is the second biological nanopore currently being investigated for DNA sequencing. A natural MspA, while favorable for DNA sequencing because of shape and diameter, has a negative core that prohibited single stranded DNA(ssDNA) translocation.  The natural nanopore was modified to improve translocation by replacing three negatively charged aspartic acids with neutral asparagines.

Co-transfection

1. Viral proteins + cDNA --> Virus --> transduction --> produce protein of interest


2. Cre/loxP recombination --> gene disturbance / addition of a gene

MALDI-TOF MS

MALDI-TOF MS:
-consist of 3 components: (i) a specimen ionization chamber, within which the laser-based vaporization of the specimen takes place;
(ii) a time of flight mass analyzer; and
(iii) a particle detector

- sample is spotted onto a target
plate, along with a chemical matrix (5).
The matrix is selected for certain prop-
erties, including easy sublimation, e.g. α-cyano-4-hydroxycinnamic acid

-sample-matrix
mixture is pulsed with a laser, Ultra-
violet nitrogen lasers (337 nm)

- Laser irradi-
ation results in vibrational excitation of
the matrix and the ejection (desorption)
of analyte molecules surrounded by
clusters of matrix molecules, water, and
ions. Once desorbed, the matrix mole-
cules transfer protons to the analyte,
resulting in positively charged analyte
cations in the gas phase.

-accelerated
across an electric field within the ion-
ization chamber to a velocity that
depends on the mass-to-charge (m/z)
ratio of the analyte.

New Role in TB testing

Quantiferon-TB assay (QFT):

Collect blood-->simulate cells with purified protein derivative (PPD)-->detect IFN-gamma by enzyme immunoassay (EIA), which serve as an indirect indicator of exposure to TB

QFT Gold (QFT-G):
-replaced the PPD antigen with early secreoty antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10)
-phytohemagglutinin (PHA) as a +ve control, a mitogen to ensure cells are responding to antigens
-+ve control must show a production of IFN-gamma >=0.5IU/ml
-positive immune response to TB if IFN-gamma >=0.35IU/mi after substracting the value of the Nil control
-IFN-gamma <0.35IU/ml and IFN-gamma of +ve control >=0.5IU/ml ==> unlikely to have TB infection



The manufacturer’s package insert states that a positive QFT result does not “necessarily indicate the presence or absence of active tuberculosis disease.” Other diagnostic procedures, such as mycobacterial culture and radiologic examination, should be used when there is clinical suspicion of TB disease.

Sulfonylurea

Sulfonylurea (UK: sulphonylurea) derivatives are a class of antidiabetic drugs that are used in the management of diabetes mellitus type 2. They act by increasing insulin release from the beta cells in the pancreas.

Mechanism of action

Sulfonylureas bind to an ATP-dependent K⁺(K_ATP) channel on the cell membrane of pancreatic beta cells. This inhibits a tonic, hyperpolarizing efflux of potassium, thus causing the electric potential over the membrane to become more positive. This depolarization opens voltage-gated Ca²⁺ channels. The rise in intracellular calcium leads to increased fusion of insulin granulae with the cell membrane, and therefore increased secretion of (pro)insulin.

Knudson hypothesis

PCR allele dropout

there is mutation exists at primer-binding site,
primer cannot bind firmly,
leading to one peak in DNA gel.