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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
  • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

• the present invention is in the field of molecular biology and diagnostics, and relates in particular to an improved method of detecting the presence of nucleotide sequences, e.g. point mutations, within a double-stranded DNA.
• the method is useful, for example, in the early detection of lung and colon cancer.
• Cancer is the second overall leading cause of death, after ischemic heart disease, in the United States and Western Europe and despite recent advances in its treatment, there is, for most cancer types, no miracle cure on the horizon. Cancer causes approximately 25 % of all deaths. The incidence continues to rise, probably reflecting the increasing average age of the population. The key to survival is early diagnosis and treatment.
• Lung cancer has a high incidence and mortality. Early detection programs with conventional methods such as X-ray and sputum cytology have failed to improve mortality. Lung carcinomas are now considered a genetic disease. Many regions in the genome have been thought to contain candidate genes related to the development of lung cancer. The mutations in p53 and Kirsten ras (K-ras) genes are the best characterized in lung cancer and are thought to occur late in the development of lung cancer. Therefore, new approaches that use genetic alterations such as K-ras as potential biomarkers may be beneficial for early detection of lung cancer (Somers V.A.M.C, Thesis Maastricht University (1998), Netherlands). Point mutations in the human genome play a central role in tumorigenesis
• this method is based on the following principle: after exonuclease digestion, polymerase chain reaction fragments are determined by simultaneous hybridization with a capture probe and a detection probe complementary to sequences near the 3' end of the antisense fragment.
• the capture probe bears a biotin residue and the detection probe digoxigenin.
• the PCR product hybrids are captured in streptavidin-coated microtiter plates and detected with labeled anti-digoxigenin antibody. For the detection of known point mutations this procedure was extended by using after the capture step the ligation of a mutation-specific capture probe with adjacent detection probe ("Point-EXACCT").
• Point-EXACCT Essential in the Point-EXACCT technique is that only molecules will be detected by this format which have been hybridized with two probes and subsequently ligated, resulting in a very high degree of specificity.
• Point-EXACCT requires considerably less time and effort as compared to other techniques used for the detection of known point mutations. The method can be easily automated, permitting rapid screening of tissue banks with multiple probes to individual base substitutions, deletions or additions.
• K-ras oncogene Point mutations in the K-ras oncogene are one of the most common genetic alterations involved in various types of human cancer. In lung cancer, K-ras mutations occur predominantly in codon 12. The frequency of those alterations varies within different histological subtypes. K-ras point mutations are found in approximately 15-56% of the adenocarcinomas and to a lesser extent in other types of non-small cell lung carcinomas (NSCLC). K-ras point mutations at codon 12 are found to occur early during lung cancer development. See Somers V.A.M.C, Thesis ibid., and references mentioned therein. Somers V.A.M.C, et al., Clinical Chemistry (1998) compared the detection of
• Point-EXACCT K-ras point mutations by Point-EXACCT with direct sequencing of double and single- stranded amplification products.
• Point-EXACCT showed clear advantages as compared to previously described highly sensitive methods.
• the Point-EXACCT has been designed for analysis of single base substitutions, where the exact place of said substitutions in the nucleotide sequence of the gene to be analyzed is known beforehand.
• Validation of the method for the detection of known point mutations in a large group of patients with NSCLC has confirmed its high sensitivity.
• a relatively low amount of target cells is required before a signal is obtained.
• the ligation step is crucial. The whole procedure is laborious, and therefore there is a clear need for an accurate and more efficient method.
• nucleotides are either synthesized or spotted on the glass slides.
• oligonucleotides are built by light-directed sequential oligonucleotide synthesis on the solid support; see U.S. Patent No. 5,925,525 and WO 97/27317.
• Light is used to activate a predetermined site for the chemical coupling of a nucleotide.
• An array of nucleotides is built in successive rounds of this site-directed light- activated building process.
• oligonucleotides are synthesized separately in an individual fashion and later bound to the solid support with NH 2 linkage.
• the solid support is coated, e.g. with silane or poly-L-lysine, and a robotic arrayer is used for printing. This robot arrayer may be based on different principles, such as piezoelectric printing, and the ring and pin mechanism.
• ligase in microarray analysis has been mentioned in WO 97/31256 and US 5,242,794 as a way for detection of one or more of a plurality of sequences differing by one or more single base changes, insertions, deletions or translocations in a plurality of target nucleotide sequences.
• the ligase step in the procedure is similar to the one mentioned for the Point-EXACCT procedure, except that exonuclease is not a part of the procedure.
• a filter is used as solid support in US 5,242,794 a filter is used.
• the microarray technique allows large scale nucleic acid analysis, but require a large amount of target cells, since the detection mechanism is based on hybridization. Hence there is a need for optimization.
• the present invention provides a further improvement of the point-EXACCT method using the microarray technique, wherein the benefits of both methods are successfully combined.
• a method of detecting the presence of a nucleotide sequence within a double-stranded DNA in a sample comprising the following steps: a. coating a solid support with a first layer of biotinylated serum albumin, and a second layer of streptavidin molecules having sufficient density to perform efficient microarray analysis; b. digesting the double-stranded DNA with an exonuclease to convert double- stranded to single stranded DNA, derived from a mixture of target cells and other cells, to a single-stranded DNA; c. capturing a first nucleic acid probe adapted by biotin to said coated solid support defined in step a.; d.
• step d (ii) is adapted with the use of a mixture of partly randomized probes to allow detection of mutations without knowing the site and type of mutation beforehand.
• the capturing step is performed by capturing the first nucleic acid probe to a solid support wherein this solid support preferably is of glass, most preferably Starfrost glass.
• the support is homogeneously coated by biotinylated serum albumin and streptavidin.
• the first nucleic acid probe is printed on the solid support.
• the first nucleic acid probe is built on the solid support by light-directed oligonucleotide synthesis which is subsequently used for the remaining of the point-EXACCT procedure including the ligase step.
• the detectable moiety on the second nucleic acid probe is digoxigenin
• the detecting step is performed by binding the digoxigenin with anti-digoxigenin antibody fragments.
• the method is suitable for detecting the presence of a point mutation within the double-stranded DNA, wherein the first nucleic acid probe contains a nucleotide complementary to the nucleotide of the point mutation at the 3' end and adapted with a moiety at the 5' end which can be captured to a solid support, and the second nucleic acid probe is labeled with a detectable moiety at the 3' end which can hybridize with the single-stranded DNA adjacent the hybridized first nucleic acid probe, and wherein after ligation and denaturation the presence of captured detectable moiety indicates the presence of the point mutation within the double-stranded DNA in the sample.
• the method is suitable for detecting the presence of a point mutation within the double-stranded DNA, wherein the first nucleic acid probe contains a nucleotide at a first end complementary to the nucleotide of the wild type nucleic acid sequence at a nucleotide position suspected to be a point mutation and adapted with a moiety which can be captured to a solid support, and the second nucleic acid probe is labeled with a detectable moiety which can hybridize to the second end with the single-stranded DNA adjacent the hybridized first end of the first nucleic acid probe, and wherein the absence of captured detectable moiety indicates the presence of the point mutation within the double-stranded DNA in the sample.
• a device comprising a solid support, preferably of glass, most preferably Starfrost glass, which is suitable for carrying out the detection methods according to the present invention.
• a solid support preferably of glass, most preferably Starfrost glass, which is suitable for carrying out the detection methods according to the present invention.
• said support is coated with biotinylated serum albumin and streptavidin.
• a kit comprising a device suitable for carrying out the detection methods according to the present invention with a solid support containing a series of first nucleic acid probes.
• a kit comprising a device suitable for carrying out the detection methods according to the present invention with a solid support containing a duplicate series of first nucleic acid probes with arrangement for rapid detection.
• a kit comprising a device suitable for carrying out detection methods according to the present invention allowing serial analysis of multiple target cells on the same support.
• a kit comprising a device suitable for carrying out the detection methods according to the present invention, optionally an exonuclease, a first nucleic acid probe which binds to target DNA and which is adapted with a capture moiety, a second nucleic acid probe which binds to target DNA adjacent the first probe and which is labeled with a detectable moiety, optionally a ligase, a first antibody that binds to the detectable moiety of the second probe, a second antibody that binds to the first antibody and is labeled with an enzyme for color reaction or labeled with a fluorochrome, and a chromogen.
• a method for organizing microarray analysis on a solid support for rapid visual detection of abnormalities which comprises arranging a duplicate set of probes where the first series of arrays are for the wild-type mutation order and the second series of arrays are for the classical sequencing order.
• Figure 1 depicts a glass slide which is suitable for use as a glass support for the detection methods of the present invention.
• Squares were manually drawn on the slides, preferably having an area of about 0.25 cm 2 , with a pap pen in order to keep the fluid on this square.
• Eight squares are drawn on one slide, four for hybridization and four for ligation and denaturation.
• Figure 2 depicts a possible order for duplicate nucleotide signal, allowing rapid visual analysis.
• Figure 3 shows the wild type signal for consecutive series of nucleotides.
• Figure 4 shows besides the wild type signal background signals from the three remaining probes.
• Figure 5 shows a possible signal distribution for point mutation in a sample with target fraction.
• Figure 6 shows a possible signal distribution for a small deletion or insertion in a sample with high target fraction.
• Figure 7 shows a possible signal distribution for a point mutation in a sample with a low amount of target cells.
• Figure 8 shows a possible signal distribution in case of heterozygosity.
• Figure 9 shows a possible signal distribution for loss of heterozygosity for single nucleotide polymorphism in a sample with high target fraction.
• Figure 10 shows a possible signal distribution for loss of heterozygosity for single nucleotide polymorphism in a sample with low fraction of target cells.
• the present invention provides a significant improvement of the method of detecting the presence of a nucleotide sequence and preferably a point mutation within a double-stranded DNA in a sample, which is disclosed and claimed in U.S. Patent No. 5,744,306, the disclosure of which is incorporated herein by reference.
• the improvement is based to a great extent on replacing in the "Point EXACCT" detection method the microtiter plates as solid supports by microarray technology which involves the use of solid e.g. glass supports.
• microarray' or 'chip' technique or technology are synonyms and are meant to indicate analysis of a plurality of small spots of nucleic acids distributed on a small surface area to facilitate large scale nucleic acid analysis enabling the simultaneous analysis of thousands of DNA and/or RNA sequences.
• the terms are likewise applicable to the analysis of peptides or proteins in a similar way.
• microarray technology can be successfully applied on the Point-EXACCT detection method, replacing microtiter plate format which uses a volume of about 100 ⁇ l per well with streptavidin coated glass slides using a volume of 120 ⁇ l for all spots on the support, and thus enabling to perform Point- EXACCT with smaller samples and at much larger scale of operation.
• the detection mode may vary but the invention can be suitably carried out with absorption microscopy and other modes, such as fluoresence and laser scanning microscopy, which will be evident to a person skilled in the art.
• absorption microscopy and other modes such as fluoresence and laser scanning microscopy, which will be evident to a person skilled in the art.
• glass slides in particular starfrost glass slides, but other materials such as silica or glass slides of other types may also give satisfactory results. This can easily be determined by a person skilled in the art.
• the glass slides preferably contain squares drawn on the glass in order to keep the fluid on this square.
• the size of the squares is not critical. A preferred size has an area of about 0.25 cm 2 , so that 8 squares can be drawn on 1 glass slide, four for hybridization and four for ligation and denaturation. This is convenient for testing one base on a slide.
• the slides normally are coated by conventional techniques which include coating with biotinylated serum albumin (usually BSA) which is soaked by streptavidin. Excellent results are obtained when a first homogeneous coating of biotinylated BSA is applied which after drying is incubated by streptavidin. A high concentration of biotinylated albumin is necessary to get sufficient binding sites for the capture probes. In the concentrations used, the effect of the surface tension generally will result in an inhomogeneous protein distribution. Homogenation is obtained by additional measures before drying, for example covering the protein layer with non-adherent weight or using a surfactant. In an embodiment of the present invention the coating allows high density deposition by robots of biotin labeled substances such as nucleic acids and proteins.
• the coated supports can be stored for long periods of time (up to 12 months) at room temperature.
• the surface area of the coated support is not critical and may vary according to the wishes of the user. Usually it will be up to about 10x10 cm 2 .
• Slides, preferably glass slides, and more preferably Starfrost glass slides of about 7.5x2.5 cm are convenient and give satisfactory results, and are therefore preferred.
• the coating procedure descibed above which results in a homogeneous coating layer is simple and the capture probes are linked neither chemically nor with UV radiation to the glass surface.
• a homogeneous layer of biotin-labeled substances such as nucleic acids and proteins can be suitably used for microarray analysis.
• the array technique on the Point-EXACCT detection method according to the invention worked well with high concentrations of all products. Once the principle was established, the concentrations were brought back step by step to fmol-pmol range. Thus, the concentrations of the products to be detected and the reagents used in the modified Point-EXACCT method according to the invention usually differ from those applied in the conventional Point-EXACCT method and can be determined and optimized by routine experimentation. As appears from the Experimental Part hereinbelow, suitable and preferred concentrations are as follows:
• biotinylated capture probes about 5 ng/0.15 ⁇ l of biotinylated probe - for the digoxigenin-labeled capture probe: about 5 ng/0.15 ⁇ l of digoxigenin-labeled probe
• - for the concentration of anti-digoxigenin antibody about 5 ⁇ l with a concentration of about 1 ⁇ g/ml - for the concentration of rabbit anti-mouse labeled with alkaline phosphatase: about 2.25 x 10 "7 g/5 ⁇ l.
• a variety of tissue samples was tested with the modified Point-EXACCT method involving the array technology, including wild types and samples which were known to contain certain K-ras mutations, which were predetermined by the conventional Point-EXACCT procedure.
• the results of the conventional Point-EXACCT and the microarray approach were identical, the same mutations were found with both tests.
• a vertical slide holder is used comprising a small fluid-containing incubation 'chamber'.
• One of the broad vertical walls of the incubation chamber consists of the solid support with the microarrays (about 5x2 cm). The distance between both broad vertical walls is small, e.g.
• Another embodiment of the invention is the parallel hybridization in the vertical mode of two solid supports with the same capture probe composition.
• the first support is used for quantification of the target DNA without ligation and denaturing step (also called hybridization only), whereas the second support is used for quantification after ligation, denaturing, and staining.
• the ratio between the signals of the corresponding capture probe determines the outcome. This ratio is obtained by dividing the signal of the support 'without ligation and denaturation' by the signal of the support 'with ligation and denaturation'.
• This setup requires high quality of coating of the supports, and of the spotting steps, emphasizing the relevance of the coating according to the present invention. All other steps are performed in parallel by robotic and do not give rise to any essential variations.
• the present invention of the Point-EXACCT microarray method uses beside two hybridizations the ligase reaction for signal discrimination without significant loss of signal intensity. This has the advantage that also a lower amount of target cells can be present in the biological sample. While in the conventional microarray technology from 100% to 10% of the sample material has to be hybridized in order to obtain a reliable signal discrimination, this range is extended in the method of the present invention to from 100% to about 0.1 %.
• the subsequent ligase step according to the present invention has certain distinct advantages. Identical hybridization conditions can be used in both instances. Consequently, discrimination between matches and mismatches is very high, much higher than can be achieved with any other methods, such as oligonucleotide mutation specific hybridization wherein the effects of GC content were only somewhat neutralized in high concentrations of quarternary or tertiary amines.
• the signals of the well 'without ligation and denaturation' and the signal of the well 'with ligation and denaturation' are in close approximation to each other
• these signals are preferably obtained by different supports. This allows the calculation of the ratio as mentioned above.
• An embodiment of the invention is that with the microarray format the support 'with the ligation and denaturation' steps is sufficient in itself to acquire the information about the presence of mutation. Since information about the wild type signal can be obtained from a series of contiguous nucleotide positions, a normal range with its distribution (i.e. without mutations) can be calculated for each support.
• the hybridization signal in the support that is used for 'hybridization only' should be high as well and serves and quality control for hybridization and spotting.
• Another capture probe design useful for known single nucleotide polymorphism analysis is the presence of only two capture probes on the solid support. For a single nucleotide polymorphism locus either homozygosity for one of the two possibilities, i.e. probes or heterozygosity is present. After Point-EXACCT 'with ligation and denaturation' only a high signal will be present for the homozygous nucleotide. In contrast, in case of heterozygosity both probes will show a clear signal. However, this signal amount is lower than for homozygosity.
• Ratio calculations provide clear distinction between homozygosity and heterozygosity. This part of the invention works satisfactory on blood samples containing white blood cells with a fairly homogeneous composition of DNA.
• Another design of probe deposition according to the invention is the presence of duplicate series of capture probes with a different arrangement. In the sequence type of arrangement, the nucleotides usually are positioned in four arrays where the ends of the capture probe on the 3' site will be the same. Thus, one array will end on adenine (A nucleotide), the others on guanine (G nucleotide), thymine (T nucleotide) and cytosine (C nucleotide), respectively.
• the combination of two different capture probes arrangements is useful.
• the second arrangement is suitably based herein on wild type option in the first arrangement and the three other nucleotide options are then deposited on the next three arrays. If no mutations are present, only the wild type array should have high signals with Point-EXACCT. Any change from normal is an indication of a mutation.
• this capture probe arrangement is that any deviation from a long line of positive signals is detected with the naked eye in a split second.
• the analysis according to the method of this invention is very rapid.
• the wild type arrangement can be used for rapid detection of the mutated region and the adjacent sequence order to determine or get an approximation of the type of mutation.
• one lane is used for marking, the next four lanes are used for the wild-type arrangement of the probes, and the next four lanes for sequencing arrangement.
• the mark lane can also be used to depict the single nucleotide polymorphic loci.
• loss of heterozygosity is detected applying the characteristic of heterozygosity for single nucleotide polymorphism with the modified Point-EXACCT method according to the invention.
• Loss of an allele frequently occurs in tumor cells. If the biological sample contains a high proportion of tumor DNA, such as DNA from a tumor cell line or laser dissected tumor cells, and a part of the allele containing the single nucleotide polymorphism is lost, then only one of the two probes will be positive with Point-EXACCT. This can be detected by comparison with another sample of the patient containing wild type DNA, e.g. from a blood sample or buckal smear.
• Point-EXACCT Previously mentioned detection procedures with Point-EXACCT require a prior knowledge about the nucleic acid sequence to be analyzed for example detection of a specific point-mutation, single nucleotide polymorphism, or loss of such a polymorphism. With the microarray Point-EXACCT technique mutations can also be detected which are not known beforehand. In malignant tumor development some genes, especially tumor suppressor genes are known for a mutation prone region.
• the second oligonucleotide with detection moiety is specific for a certain genomic region.
• the second labeled oligo- nucleotide may contain nonspecific nucleotides, such as inosine or others, in order to reduce the number of second oligo probes for a more general application. More specifically, a combination of 'partly random probes' may be useful.
• the so called 'partly random probes' are oligomers of about 12 or preferably less 9 or more preferably 6 nucleotides long and designed in the following way. On each position only the same 3 of the 4 possible oligonucleotides are randomly incorporated. A mixture of the possible four different oligonucleotides results in a high number of possible random probes.
• the mixture consists of (GAT)n, (GAC)n, (GCT)n and (ACT)n, where n is the length number of the oligonucleotide, e.g. 9, 8, etc.
• the 3' end of these probes is labeled with digoxigenin or other labels (see below).
• digoxigenin or other labels see below.
• the 5' end of these oligomers first needs to be chemically phosphorylated.
• incorporation of one or more non-specific nucleotides at any of nucleotide positions is useful.
• T pyrimidine thymine
• U uracil
• One option is the design with capture probes aligned with the four possible options for each DNA RNA base, representing an array with a known part of the genome. If after the hybridisation of target sequences and detection probes, ligation and staining procedure just the wild type capture probes are visualised the normal sequence is present only . However, if (part of) the other capture probes are visible, this denotes the presence of possible point mutation(s), deletion(s) and/or insertion(s), provided that the range of deletion(s) / insertion(s) is at least partly covered by the array of capture probes.
• the new modified Point-EXACCT method according to the invention involving the microarray technology is fast (more than 1000 nucleotides in 200 specimen can be analyzed in one day), highly specific and highly sensitive, relatively cheap and it can be automated.
• the method is also user-friendly, i.e. the convenient solid open format of the microscope slide, the non-radioactive, non-toxic, low-volume hybridization solution, and the comforting knowledge that the arrays are cheap and easily replaceable.
• the transfer to a smaller scale has many advantages, including working with smaller samples (fmol/pmol level), whereby lots of samples can be processed at the same time. Ten thousand probes can be spotted on one glass slide which enables the use of the modified Point-EXACCT method for large scale testing, e.g. simultaneous testing of a plurality of sequences from one gene or different genes from one or more patients.
• the modified Point-EXACCT method according to the invention is also suitable for large scale screening of point mutations, insertions, deletions, and loss of heterozygosity at the same time.
• the method of the present invention involving the array technology also offers a plurality of possibilities for further improvement.
• the biotin labeled substance can serve as capture probe for subsequent binding of nucleic acids (NA)/protein.
• NA/Proteins of interest, NPOI nucleic acids of interest
• the NPOI in turn can be made visible by direct labeling during synthesis of NPOI before capturing or, in case of unlabeled NPOI, by subsequent binding of labeled detection probe.
• a fluorescence mode is used for reasons of higher sensitivity, larger dynamic range, instead of extinction mode.
• the latter method has the advantage that the outcome can be made visible with regular light microscopy.
• microorganisms e.g. for clinical and food applications, such as human papilloma virus, legionella, tuberculosis, etc.
• the modified Point-EXACCT method according to the invention is typically described herein using alkaline phosphatase for detection with absorption microscopy.
• this method can be replaced by different labels, including radio nucleotides, substrates, magnetic particles, heavy metal atoms, and particular fluorescers, chemiluminescers and spectroscopic labels, other enzymes than alkaline phosphatase, etc., which may change the sensitivity and/or the dynamic range of the system.
• the detection system best adapted for high resolution and high sensitivity detection may be selected.
• this may be fluorescence or chemiluminescence optical systems.
• Other detection systems may be adapted to the purpose, e.g. IR microscopy, atomic force microscopy, electrical conductance, image plate transfer, and interference microscopy (e.g. Jamin-Lebedief).
• IR microscopy e.g. IR microscopy
• atomic force microscopy e.g. atomic force microscopy
• electrical conductance e.g.
• image plate transfer e.g. Jamin-Lebedief
• a plurality of materials was tested. Among these were glue-coated, silane- coated, uncoated, poly-L-lysine-coated, vectabond-coated, APTS-BSA-coated, starfrost, superfrost and superfrost+ glass slides.
• the coating of biotinylated bovine serum albumin and streptavidin gave the best results on starfrost glass slides and, consequently, this material was selected for further experiments.
• bio-BSA biotinylated bovine serum albumin
• the parafilm may be covered with a small weight, e.g. another glass slide.
• the length of the linker between biotin and nucleotides may be of variable length, 4-16 carbon atoms give satisfactory results
• M A-D mouse-anti-digoxigenin antibody
• Point-EXACCT in vertical mode on starfrost glass slides with spotted biotinylated probes The purpose of this procedure was to perform Point-EXACCT in a vertical mode with a pipetting robot (e.g. Mark 5) Such a robot can be used for automatic immunostainings on histologic and cytologic specimens.
• a pipetting robot e.g. Mark 5
• the biotinylated probes were spotted with a microaray robot on the coated glass slides.
• the protocols are schematically given below.
• bio-BSA biotinylated bovine serum albumin
• GMS Genetic Microsystems
• Mark 5 needs 120 ⁇ l to fill the vertical holder, from which 40 ⁇ l is needed to rinse the previous liquid and the remaining part to fill the incubation space with liquid. As compared to the original procedure, only half of the volume was needed, since through the capillary activity, the 60 ⁇ l product stays in the upper half of the holder where the probes are spotted.
• the concentration biotinylated bovine serum albumin from 1 mg/ml to 2 ⁇ g/ml was varied. The rest of the settings remained standard according to the maximum protocol.
• Table 1 shows the results for the hybridization and ligation signals. A semi- quantitative score of the staining is provided.
• the hybridization signals gave a strong staining up to a concentration of 2 ⁇ g/ml.
• the staining of the ligation signal of the G-base decreased after a concentration of about 16 ⁇ g/ml. Therefore a concentration of 20 ⁇ g/ml was applied in further experiments. From this concentration, 5 ⁇ l was used for a square of 0.25 cm 2 . Thus, for this square 100 ng biotinylated bovine serum albumin was used which corresponds with 8.8 x 10 11 molecules/square or 1.46 x 10 "12 mol.
• the streptavidin concentration was varied in a range from 500 ⁇ g/ml to 1 ⁇ g/ml.
• the optimum concentration 20 ng/ ⁇ l. was used. The rest of the settings remained standard according to the maximum protocol.
• the optimal streptavidin concentration appeared to be 40 ng/ ⁇ l. This was used in subsequent experiments.
• a similar setup was used and optimal concentration for concentration biotinylated probes appeared to be 5 ng/0.15 ⁇ l will be used. This equals approximately 3.9 x 10 11 molecules in a spot of 150 micron diameter.
• the optimal concentration of the dig-labeled probes appeared to be of 5 ng/0.15 ⁇ l. This 5 ng contained the same amount of molecules and mol as the biotinylated capture probes.
• the minimum amount of PCR product was 1 ⁇ g was needed for a square of 0.25 cm 2 . This corresponds to approximately 10 10"14 copies.
• the optimal concentration of the first mouse-anti-digoxigenin antibody was 1 ⁇ g/ml. This corresponded to 2 x 10 11 molecules and 3.3 x 10 "13 mol per square.
• the optimal concentration of rabbit anti-mouse labeled with alkaline phosphatase was acquired with 45 mg/l RAM-AP. This equals 2.25 x 10 ⁇ 7 g/5 ⁇ l used, which means 9 x 10 12 molecules or 1.5 x 10 "11 mol. All experiments were carried out with a horizontally positioned solid support and hybridization and washing steps were performed by adding drops and sucking it off individually.

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