Sensitive and specific colorimetric DNA detection by invasive reaction coupled with nicking endonuclease-assisted nanoparticles amplification.

Colorimetric DNA detection is preferable to methods in clinical molecular diagnostics, because no expensive equipment is required. Although many gold nanoparticle-based colorimetric DNA detection strategies have been developed to analyze DNA sequences of interest, few of them can detect somatic mutations due to their insufficient specificity. In this study, we proposed a colorimetric DNA detection method by coupling invasive reaction with nicking endonuclease-assisted nanoparticles amplification (IR-NEANA). A target DNA firstly produces many flaps by invasive reaction. Then the flaps are converted to targets of nicking reaction-assisted nanoparticles amplification by ligation reaction to produce the color change of AuNPs, which can be observed by naked eyes. The detection limit of IR-NEANA was determined as 1pM. Most importantly, the specificity of the method is high enough to pick up as low as 1% mutant from a large amount of wild-type DNA backgrounds. The EGFR gene mutated at c.2573 T>G in 9 tissue samples from non-small cell lung cancer patients were successfully detected by using IR-NEANA, suggesting that our proposed method can be used to detect somatic mutations in biological samples.

Improvement of LATE-PCR to allow single-cell analysis by pyrosequencing.

Nucleic acid analysis in a single cell is very important, but the extremely small amount of template in a single cell requires a detection method more sensitive than the conventional method. In this paper, we describe a novel assay allowing a single cell genotyping by coupling improved linear-after-the-exponential-PCR (imLATE-PCR) on a modified glass slide with highly sensitive pyrosequencing. Due to the significantly increased yield of ssDNA in imLATE-PCR amplicons, it is possible to employ pyrosequencing to sequence the products from 1 µL chip PCR which directly used a single cell as the starting material. As a proof-of-concept, the 1555A>G mutation (related to inherited deafness) on mitochondrial DNA and the SNP 2731C>T of the BRCA1 gene on genomic DNA from a single cell were successfully detected, indicating that our single-cell-pyrosequencing method has high sensitivity, simple operation and is low cost. The approach has promise to be of efficient usage in the fields of diagnosis of genetic disease from a single cell, for example, preimplantation genetic diagnosis (PGD).

Digital detection of multiple minority mutants in stool DNA for noninvasive colorectal cancer diagnosis.

Somatic mutations in stool DNA are quite specific to colorectal cancer (CRC), but a method being able to detect the extraordinarily low amounts of mutants is challengeable in sensitivity. We proposed a hydrogel bead-array to digitally count CRC-specific mutants in stool at a low cost. At first, multiplex amplification of targets containing multiple mutation loci of interest is carried out by a target enriched multiplex PCR (Tem-PCR), yielding the templates qualified for emulsion PCR (emPCR). Then, after immobilizing the beads from emPCR on a glass surface, the incorporation of Cy3-dUTP into the mutant-specific probes, which are specifically hybridized with the amplified beads from emPCR, is used to color the beads coated with mutants. As all amplified beads are hybridized with the Cy5-labeled universal probe, a mutation rate is readily obtained by digitally counting the beads with different colors (yellow and red). A high specificity of the method is achieved by removing the mismatched probes in a bead-array with electrophoresis. The approach has been used to simultaneously detect 8 mutation loci within the APC, TP53, and KRAS genes in stools from eight CRC patients, and 50% of CRC patients were positively diagnosed; therefore, our method can be a potential tool for the noninvasive diagnosis of CRC.

Multiplex loop-mediated isothermal amplification detection by sequence-based barcodes coupled with nicking endonuclease-mediated pyrosequencing.

The loop-mediated isothermal amplification (LAMP) is a well-developed method for replicating a targeted DNA sequence with a high specificity, but multiplex LAMP detection is difficult because LAMP amplicons are very complicated in structure. To allow simultaneous detection of multiple LAMP products, a series of target-specific barcodes were designed and tagged in LAMP amplicons by FIP primers. The targeted barcodes were decoded by pyrosequencing on nicked LAMP amplicons. To enable the nicking reaction to occur just near the barcode regions, the recognition sequence of the nicking endonuclease (NEase) was also introduced into the FIP primer. After the nicking reaction, pyrosequencing started at the nicked 3' end when the added deoxyribonucleoside triphosphate (dNTP) was complementary to the non-nicked strand. To efficiently encode multiple targets, the barcodes were designed with a reporter base and two stuffer bases, so that the decoding of a target-specific barcode only required a single peak in a pyrogram. We have successfully detected the four kinds of pathogens including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum (TP), which are easily infected in blood, by a 4-plex LAMP in a single tube, indicating that barcoded LAMP coupled with NEase-mediated pyrosequencing is a simple, rapid, and reliable way in multiple target identification.

Digital analysis of the expression levels of multiple colorectal cancer-related genes by multiplexed digital-PCR coupled with hydrogel bead-array.

To digitally analyze expression levels of multiple genes in one reaction, we proposed a method termed as 'MDHB' (Multiplexed Digital-PCR coupled with Hydrogel Bead-array). The template for bead-based emulsion PCR (emPCR) was prepared by reverse transcription using sequence-tagged primers. The beads recovered from emPCR were immobilized with hydrogel to form a single-bead layer on a chip, and then decoded by gene-specific probe hybridization and Cy3-dUTP based primer extension reaction. The specificity of probe hybridization was improved by using electrophoresis to remove mismatched probes on the bead's surface. The number of positive beads reflects the abundance of expressed genes; the expression levels of target genes were normalized to a housekeeping gene and expressed as the number ratio of green beads to red beads. The discrimination limit of MDHB is 0.1% (i.e., one target molecule from 1000 background molecules), and the sensitivity of the method is below 100 cells when using the ß-actin gene as the detection target. We have successfully employed MDHB to detect the relative expression levels of four colorectal cancer (CRC)-related genes (c-myc, COX-2, MMP7, and DPEP1) in 8 tissue samples and 9 stool samples from CRC patients, giving the detection rates of 100% and 77%, respectively. The results suggest that MDHB could be a potential tool for early non-invasive diagnosis of CRC.

Highly sensitive pyrosequencing based on the capture of free adenosine 5' phosphosulfate with adenosine triphosphate sulfurylase.

In pyrosequencing chemistry, four cascade enzymatic reactions with the catalysis of polymerase, adenosine triphosphate (ATP) sulfurylase, luciferase, and apyrase are employed. The sensitivity of pyrosequencing mainly depends on the concentration of luciferase which catalyzes a photoemission reaction. However, the side-reaction of adenosine 5' phosphosulfate (APS, an analogue of ATP) with luciferase resulted in an unavoidable background signal; hence, the sensitivity cannot be much higher due to the simultaneous increase of the background signal when a larger amount of luciferase is used. In this study, we demonstrated a sensitive pyrosequencing using a large amount of ATP sulfurylase to lower the concentration of free APS in the pyrosequencing mixture. As the complex of ATP sulfurylase and APS does not react with luciferase, a large amount of luciferase can be used to achieve a sensitive pyrosequencing reaction. This sensitivity-improving pyrosequencing chemistry allows the use of an inexpensive light sensor photodiode array for constructing a portable pyrosequencer, a potential tool in a point-of-care test (POCT).

Gene expression analysis on a photodiode array-based bioluminescence analyzer by using sensitivity-improved SRPP.

Most methods used for gene expression analysis are based on dye-labeling, which requires costly instruments. Recently a dye-free gene expression analysis method-SRPP (Sequence-tagged reverse-transcription polymerase chain reaction coupled with pyrosequencing) was developed to compare relative gene expression levels in different tissues, but the throughput of the SRPP assay is very limited due to the use of a photomultiplier tube (PMT)-based pyrosequencer for the detection. To increase the throughput of the SRPP assay, an inexpensive photodiode (PD) array-based bioluminescence analyzer (termed as "PD-based pyrosequencer") was coupled to SRPP; however the low sensitivity of PD limited the wide application of SRPP. To enable SRPP analyzing low abundance genes in clinical samples, sequence-tagged gene-specific primers instead of sequence-tagged poly (T)(n) primers were used for reverse-transcription, and the SRPP sensitivity was thus improved more than 10 times. This improvement compensates the sensitivity loss due to the use of PD in a pyrosequencer. The accurate determination of the expression levels of ten prognostic marker genes (AL080059, MMP9, EXT1, ORC6L, AF052162, C9orf30, FBXO31, IGFBP5, ESM1, and RUNDC1) differing between normal tissues and tumor tissues of breast cancer patients demonstrated that SRPP using gene-specific RT primers coupled with the PD array-based bioluminescence analyzer is reliable, inexpensive, and sensitive in gene expression analysis.

Highly sensitive mutation detection based on digital amplification coupled with hydrogel bead-array.

In order to detect small amounts of mutants for early cancer diagnosis, we have developed the novel method of using amplicon-coated microbeads and single-molecule-PCR in water-in-oil emulsions, which we coupled with a new detection platform, the hydrogel bead-array, a 3-D polyacrylamide gel network used as a carrier to immobilize the beads.