In general, the microarrays of single-stranded oligonucleotides (usually DNA or PNA) immobilized on the surface of glass or other solid surfaces are used for various purposes: identification or detection of infectious agents such as virus, bacteria, and other forms of microorganism, genotyping, disease causing genes, mutation of genes, expression of genes, etc. In some instances, the technology has also been adapted for diagnosis at hospitals and clinics. In other instances, various technologies have been consolidated and optimized for convenient use and or reliable decisions in connection with clinical diagnosis.
The use of DNA chip often requires long processes such as (a) extraction of DNA or RNA from biological specimens such as tissues, cells, blood, serum, and biological fluids, (b) amplification of target regions of genes by polymerase chain reaction (PCR), or conversion of RNA to cDNA and subsequent amplification of cDNA, (c) labeling of amplified DNA with fluorescent tags, often during PCR, (d) hybridization of the amplified DNA with the oligonucleotides (often called capture probes) immobilized on the surface of a solid substrate such as glass, and (e) scanning of the slide in a detector such as a laser scanner.
With respect to extraction, DNA or RNA is obtained from biological specimens by well established protocols (Molecular Cloning: A laboratory manual by Sambrook and Russel, 3rd Edition, Cold Spring Harbor Laboratory Press, 2001). Typically cells are lysed in a buffer containing SDS and proteinase K for degradation of proteins, the lysate is extracted with phenol/chloroform, and the DNA or RNA in the phenol layer is precipitated with ethanol.
Several companies have introduced more simplified protocols for high throughput purification of DNA and RNA from biological specimens. In general, these procedures take advantage of the binding property of DNA and RNA to silica powder, silica fiber or silica membrane (GE Heatlth, Qiagen, etc.). These procedures, although can be accomplished in 30 minutes to one hour, requires multiple pipetting, and many manipulations.
As an example, one protocol is briefly described. Biological specimens such as cells are suspended in 200 μl PBS (phosphate buffered saline) and centrifuged (300 g for 5 min). To the pelleted cells 200 μl lysis buffer and 20 μl proteinase K is added. The mixture is incubated at 70° C. for 10 min. Ethanol (200 μl) is added and the tube is vortexed. The lysed cells are transferred into the Mini spin column (with a silica membrane bottom) placed into a 2 ml collection tube and centrifuged at 6000×g for 1 minute and the collection tube is discarded. The Mini-spin column is transferred to another collection tube, 500 μl of wash buffer is added to the Mini spin column and centrifuged. The collection tube is discarded and the Mini-spin column is transferred to another collection tube. A second wash buffer (500 μl) is added and the tube is centrifuged. The Minispin column is transferred to another collection tube, and 200 μl of elution buffer is added to the mini-spin column followed by centrifugation. The collection tube is saved. The Minispin column is then transferred to another collection tube and second elution buffer is added to the Mini-spin column and centrifuged again. The collection tube is saved. The DNA in two collection tubes are combined. As seen from this example, extraction of DNA from cells requires about 10 pipetting steps and several centrifugation steps and many manipulations such as transfer of Mini-spin column to different collection tubes, etc. Purification of RNA also requires a similar process.
Similar protocols are adopted for extraction of nucleic acids from multiple samples in an automated format.
Often, the target DNA or RNA sequences for diagnosis are present in biological samples in a very minute amount, and there is need for amplification of target DNA or RNA sequences before nucleic acid diagnosis. One method for amplification of target DNA sequences is polymerase chain reaction (PCR). Generally, the PCR process involves denaturation of DNA (strand separation) at a high temperature (95° C.), annealing of short primers (usually 15-20 nucleotides) that are complementary to either ends of a DNA region to be amplified, and chain elongation from the annealed primers in the presence of thermostable DNA polymerase and four deoxynucleotide triphosphates. These processes require specific temperatures: for example, 90° C. or above for denaturation, 55-65° C. for annealing, and 72° C. for chain elongation. Denaturation, annealing and chain elongation are repeated continuously until desired amount of DNA is amplified, typically 20-30 cycles. Therefore, PCR requires a programmable thermocycler.
For amplification of a RNA sequence, the RNA is first converted to double-stranded cDNA in the presence of reverse transcriptase and DNA polymerase, DNA primers and four deoxynucleotide triphosphates. Subsequently, cDNA is amplified by PCR as described above. The process of cDNA synthesis and subsequent PCR may be carried out in one reaction mixture.
After PCR or cDNA synthesis and subsequent PCR, the amplified product is purified to remove the remaining primers and deoxynucleotide triphosphates. The PCR products are often identified by gel electrophoresis and staining of the amplified DNA with ethidium bromide or CYBR GREEN® dye. [spelled Sybr Green in claim 8]
For use of the amplified DNA in diagnosis by DNA chip, the amplified DNA is labeled. For example, a fluorescently labeled deoxynucleotide (Cy3 or Cy5-deoxy nucleotide triphosphate, ALEXA FLUOR®-deoxynucleotide triphosphate) is added to PCR reaction mixture. PCR primers that are labeled with fluorescent group or biotin can also be used for labeling amplified DNA. The biotin-labeled DNA can be detected by interaction with labeled streptavidin. The fluorescently labeled DNA can be detected by laser scanner.
After PCR, the labeled PCR product is purified by use of various protocols. One of which can be the protocol described above by use of silica membrane for extraction of DNA from cells and tissues.
The labeled PCR product is added to hybridization buffer. The mixture is heated to 95° C. to convert the labeled amplified DNA to single-stranded form and quickly chilled on ice and transferred on to a chamber assembled on the surface of a chip slide (usually a glass slide) that has immobilized single-stranded oligonucleotide (15-70 nucleotides) capture probes. The slide is then incubated at a temperature for annealing (such as from 40-65° C.). After hybridization, the chamber is disassembled and the slide is washed once with 1×SSC+0.1% SDS, once with 0.1×SSC+0.1% SDS and once with 1×SSC, all as non-limiting examples.
A DNA chip is typically a slide on which capture probes (short single-stranded DNA) are immobilized in a high density format. In many cases, a glass slide is coated with chemicals to attach the capture probes to the glass. The chemicals should not only attach the DNA to the glass surface but also minimize non-specific binding and signal noise. For example, the chemicals on a chip slide contain silanated, silylated, or poly-L-lysine as a source of an amine or an aldehyde group for attachment of DNA. Recently, dendrons have been developed for use in a biochip. When a chip is coated with dendrons, one can control spacing between capture probes and this also allows reduction of steric hinderance and concomitant increase in sensitivity. Korean patent 10-0383080-0000, and published U.S. Patent Application 2005/0037413 describe dendrons that provide controlled spacing as well as density of amines on dendron.
Single-stranded oligonucleotides whose length can vary from 15 to 70 nucleotides are often used as capture probes that will hybridize to the complementary strand sequences of target genes, DNA or RNA. PNA may also be used as capture probes. The oligonucleotides will have either amine or SH group at 3′ or 5′ ends, usually at 3′ end so that the oligonucleotides can be crosslinked to the surface of chip slide.
When DNA chip slides are used, the sequence of steps is to amplify the DNA (labeling the DNA with fluorescent group at the same time) and then purify the DNA. The amplified DNA is denatured by heating and added to the surface of capture probes for annealing. After hybridization, the slide is washed and the fluorescent double-stranded DNA is detected by scanning and the data are analyzed.
As evident from above, the whole process from extraction of nucleic acids, gene amplification, and subsequent hybridization of the amplified DNA with the capture probes on chip slide requires multiple processes such as many repeated transfer (pipetting), mixing of solutions, centrifugations, etc. Even when automated machine is developed for extraction of DNA and RNA, there is still need for coupling of this technology with PCR and hybridization with capture probes on chip surface.
Additionally, there is a requirement for considerable time for gene amplification and purification (as much as one day). Also, the cost of chemicals and disposable items is high. There is also loss of sample during purification of reaction products. Therefore, there is need for streamlining the cumbersome processes involved in use of gene chips to reduce losses and variation of the results.
There have been attempt to carry out PCR in a single step from biological specimens. For example, cells are directly added to PCR reaction mixture. Several companies have introduced one-step PCR mixtures that allow amplification of DNA from whole cells or blood. These mixtures contain detergent to facilitate lysis of cells, proteinase K for digestion of proteins, and often proprietary agents. The efficiency of PCR is variable.
Reverse transcription of RNA to synthesize cDNA with subsequent PCR can also be carried out in one step with whole cells. However, the efficiency again varies depending on the type of biological specimens. For example, blood and serum contains inhibitor of reverse transcriptase.
There have also been attempts to couple DNA and RNA extraction by an automated process and PCR. Several companies introduced combined procedures for automated extraction of DNA or RNA and automated PCR. Although these automated processes satisfy diagnosis of certain diseases as well as quantitation of target nucleic acid sequences, such as those of infectious biological agents, it is difficult to use PCR for identification of different variants of a virus (genotypes), changes in sequences in response to drug-resistance, and the mutations of target genes that cause diseases.
A DNA chip offers many advantages over diagnosis by PCR or by immunologic assays. With a DNA chip, it is possible to identify, all at once, many different sequences, changes of sequences by mutation, different genotypes of viruses, and expression of different genes. However, there is clear need for simplification and integration of all necessary protocols for extraction of nucleic acids, gene amplification and hybridization on chip surface in order for the DNA chip technology to be used at hospitals and clinics with increased throughput and reduction in manual labor. Current technology requires highly trained technicians, long processes, high cost and time. Even when automated systems are available, it requires at least three different systems, such as automated extraction of nucleic acids, gene amplification, and hybridization on DNA chip.
There is also an increasing need for diagnosis of disease-causing agents such as viruses and the changes in gene sequences that cause formation of tumors for personalized drug therapy or vaccination. For example, GLEEVEC® (imatinib) is highly effective for treatment of some cases of chronic myeloid leukaemia (CML) and also gastrointestinal stromal tumour (GIST). Gleevec specifically blocks ATP-binding sites on specific mutant tyrosine kinases in the responsive cancers. Therefore, it is necessary to detect the mutant tyrosine kinase genes before treatment of the cancer. Similarly, various oncogenic DNA and RNA viruses are known to induce tumor formation in humans. Prevention of these cancers, such as cervical cancer, requires early detection and treatment of pre-cancerous disease.
Papiloma viruses are a diverse group of DNA-based viruses that infect the skin and mucous membranes of humans and a variety of animals. Over 100 different human papilloma virus (HPV) types have been identified. Some HPV types may cause warts while others may cause a subclinical infection resulting in precancerous lesions. All HPVs are transmitted by skin-to-skin contact.
A group of about 30-40 HPVs is typically transmitted through sexual contact and infect the anogenital region. Some sexually transmitted HPVs (types 6 and 11) may cause genital warts.
Persistent infection with a subset of about 13 so-called “high risk” sexually transmitted HPVs, including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 (different from the ones that cause warts) may lead to the development of cervical intraepithelial neoplasis (CIN), vulvar intraepithelial neoplasia (VIN), penile intraepithelial neoplasia (PIN), and/or anal intraepithelial neoplasia (AIN). These are precancerous lesions that can progress to invasive cancer. HPV infection is a necessary factor in the development of nearly all cervical cancer (Walboomers, J M, Jacobs M V, Manos M M et al (1999) “Human papillomavirus is a necessary cause of invasive cervical cancer worldwide” J. Pathol. 189:12-9).
According to the Center for Disease Control (CDC), by the age of 50 more than 80% of the American women will have contracted at least one strain of genital HPV. All women are encouraged to get a yearly pap smear solely to detect cellular abnormalities caused by HPV. About 14,000 women in the United States are diagnosed with cervical cancer disease each year, and more than 3,900 women die in the United States each year from this disease.
Among the high risk HPV strains, type 16 and 18 are together responsible for over 65% of cervical cancer cases.
An HPV test detects certain types of HPVs, depending of the test. A method for detecting the DNA of high-risk HPVs has recently been added to the range of clinical options for cervical cancer screening. In March 2003, the US FDA approved a “hybrid-capture” test (see U.S. Pat. No. 6,228,578 B1), marketed by Digene, as a primary screening tool for detecting high-risk HPV infections that may lead to cervical cancer. This test was also approved for use as an adjunct to Pap testing, and may be ordered in response to abnormal Pap smear results.
The principle of Digene test is briefly summarized as follows. An exfoliated cervical cell sample is collected in a collection device and nucleic acids are released therefrom. A diluent containing multiple RNA probes to different HPV types is added. The mixture is incubated to allow hybridization of the RNA probes to the HPV sequences. The mixture is then combined with an immobilized antibody for DNA-RNA duplex, and complexes allowed to form. RNase is then added to digest away non-hybridized probe RNA. Labeled monoclonal anti-hybrid antibody is added to the tube. After incubation, excess RNase and conjugate is discarded and the hybrids detected.
The Digene test has some advantages: simplicity, rapidity, and quantitation of HPV. One major drawback of the Digene test appears to be difficulty of identification of the HPV type since mixtures of RNA probes for different HPV types are used. This is significant in view of the fact that there are many types of cancer-causing HPV types and current HPV vaccine will not protect humans from all types of HPV that cause cervical cancer. Therefore, the test is inadequate for complete identification of HPV type (s) in the infected cervical cells.
On Jun. 8, 2006, the FDA approved GARDASIL®, a prophylactic HPV vaccine which is marketed by Merck. The vaccine shows protection against initial infection with HPV types 16 and 18. Since the current vaccine will not protect women against all the HPV types that cause cervical cancer, it will be important for women to continue to seek Pap smear testing and other types of testing even after receiving the vaccine.
Various methods are available for identification of HPV type (genotyping). For example, a labeled HPV type-specific probe (labeled with radioisotope, fluorescence, biotin, etc) is directly hybridized with cellular DNA (usually after extraction and purification) in several formats. Liquid hybridization (such as the Digene “hybrid capture”), Southern and dot blot hybridization, and fluorescent in situ hybridization (FISH) are non-limiting examples. Gene amplification methods are also used. For example, HPV DNA is amplified by PCR by use of type-specific primers. However, this method suffers from the difficulty of identification of different types of HPVs in one experiment. The amplified DNA (usually labeled during PCR) can be used for hybridization with type-specific capture probes in dot blot, microtiter plate hybridization, or line probe assay in a similar way as DNA chip. However, these methods lack sensitivity and suffer from difficulties in data interpretation. DNA chip offers possibility of identification of many different types of HPV at once.
Recently, Biomedlab (Seoul, Korea) has offered a DNA chip that has capture probes for as many as 30 different types of HPVs on a single chip that can distinguish low-risk as well as high-risk HPVs with high specificity and sensitivity (U.S. Pat. No. 7,301,015). The use of this chip requires purification of DNA from patient cervical cells, PCR and labeling of amplified HPV DNA, and hybridization of the amplified DNA with the type-specific capture probes immobilized on glass slide surface.
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