E2.L4 - Molecular Diagnostics P2

TaqMan SNP Genotyping

Oligonucleotide probes containing WT or Mut. SNP. Probes have different fluorophores at 5’ end and quencher at 3’ end. Very short, and if it remains intact, no fluorescence occurs. For PCR fwd, rvs primers, and TaqMan probe are added. WT probe has complete base pairing for WT and Mut. probe has basepairing for Mut. SNP. The complete basepairing results in degradation of TaqMan probe, initiating fluorescence. Incomplete basepairing, displaces the probe and leaves it intact, inhibiting fluorescence. In carriers, both probes are degraded, and a mixed fluorescent signal is produced.

Real-Time PCR Diagnostics

SSCmec pathogenicity island adjacent to orfX, S. aureus. This provides methicillin resistance. PCR of sequence between orfX and SSCmec using rvs primer for orfX and fwd primer for SSCmec. If SSCmec is present, the PCR product can be detected by Cyber green.

DNA Fingerprinting Diagnostics

DNA taken, digestion with same restriction enzyme generates different sized fragments for most people. Fragments separated based on size by electrophoresis, HRP added to later detection. Fragments denatured and transferred to solid membrane (nitrocellulose). Single nucleic acid hybridization probe used to base pair on complementary sequences. Luminol to detect where basepairing occurred. Xray film to see the luminescence.

Detection of Epigenetic Markers

DNA methylation – addition of methyl group of cytosine in CpG dinucleotide, creates 5-methyl cytosine. Can detect variations in DNA methylation. Forward primer that goes up to C with G-complementary sequence at one end, and a non-complementary linker at the other end. Second primer pair terminates with A, and has a different con-complementary linker. For each CpG island, a downstream primer is created. # primer pairs for each CpG island for PCR.
Bisulfite added to deaminate C to U if it is not methylated. Methylation resists deamination. Methylated C will basepair with G-primer, while unmethylated C will basepair with A-primier. PCR will produce products with different linkers. The G-product will be detected after a fluorescent primer is bound to the linker and PCR allows for determination of fluorescence. The other linker will allow fluorescence of a different color.

Ancestry Determination

SNP’s in specific genes that correlate with specific ethnicities. Sepharose beads coated with DNA 50mers that end 1 nucleotide prior to SNP. ddGTP, ATP, CTP, TTP of different fluorescent colors added. They incorporate at complementary sequences. Beads harvested, and color used to determine ethnicity.

Antibiotic Resistance RNA Signatures

Growth in presence or absence of diff. Antibiotics. Different gene expression assessed. Few genes expressed at different levels, genes different for different antibiotics. Allowed for development of transcription profile assay.
Urine obtained from patients with UTI and incubated with antibiotic for 30 min. RNA isolated, expression levels assessed, and compared to standardized controls.

miRNA Signatures and Oncogenesis

MicroRNAs that regulate expression at translational level. Precursor is Pri-miRNA, which form stem-loop structures. Cleaved by Drosha RNAse to generate Pre-miRNA and exported to cytosol. Dicer RNAse cleaves to form miRNA.

RFP (Red Fluorescent Protein)

tetramer that is too large for use in many assays. Prevents proper protein localization.. PCR random mutagenesis produced a monomeric form, but it lacked fluorescence and had reduced phosphostability. However, fusing to the N- and C-teminus from GFP resolved both of the issues.

Luciferase and Biosensors

luxCDE act on plasma membrane of Gm- cell. Aldehyde substrate is liberated. luxAB produce LuxAB heterodimer that cleave substrate. This emits a photon. Only works in salt water. PCR isolates luxCDABE, operon cloned into plasmid, and introduced into organism capable of growing in fresh water. Used to test toxic ground water through detection of lack of fluorescence from cell growth.

Biosensors Detecting Steroid Hormones

Use of birth control leads to excretion of excess estrogen through urine into natural reservoirs. Yeast strain engineered with human estrogen receptor (hER) in chromosome. luxCDE constitutively produced from plasmid. A second plasmid controlled by estrogen response element contained luxAB. If clean ground water is present, hER is not present to bind to estrogen response element to induce luxAB. Contaminated ground water will allow LuxAB to cleave the substrate and release a photon