CN1944675A - High efficiency full genome fixing method - Google Patents
High efficiency full genome fixing method Download PDFInfo
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- CN1944675A CN1944675A CN 200610096841 CN200610096841A CN1944675A CN 1944675 A CN1944675 A CN 1944675A CN 200610096841 CN200610096841 CN 200610096841 CN 200610096841 A CN200610096841 A CN 200610096841A CN 1944675 A CN1944675 A CN 1944675A
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Abstract
The high efficiency full genome fixing method relates to method of amplifying full genome in high efficiency and preparing full genome DNA chip. The method includes: 1. machining pores in one flat hard material and fixing same oligonucleotide molecules; 2. cutting genome nucleic acid with enzyme into 50-1000 base size fragments and connecting the genome fragment nucleotide sequences with one pair of common connexons under the action of enzyme to form genome fragment nucleotide sequences containing connexons; 3. heat denaturing and diluting the genome fragment nucleotide sequences containing connexons and adding to the pores; and 4. adding amplifying reaction liquid into the pores and amplifying the genome fragment nucleotide sequences containing connexons to realize exponential amplification of the full genome sequence in fragment form.
Description
Technical field
The present invention relates to a kind of high efficiency whole genome amplification and complete genome DNA chip production method thereof, belong to the technical field that genome detects in the biomedicine.
Background technology
Along with going deep into of genome research, from the difference of gene level understanding life, disease takes place, the rule of development, and the interaction of medicine and life entity will become possibility.Though the factor that causes disease to take place is numerous, transgenation (single nucleotide polymorphism, methylate etc.) is widely regarded as an important internal factor.Aspect fundamental research, gene mutation site helps genomic accurate location, the genetic development of study of disease gene, clone's Disease-causing gene; In application facet, directly seek the susceptibility gene mutation site of disease: most of complex diseases, as cancer, diabetes, cardiovascular disorder, dysthymia disorders, asthma etc., be audient's polygene and environmental factor acting in conjunction, by identifying on a large scale for mutator gene type in the genome sample of a large amount of a certain specified diseases and detecting, can obtain about with the information of this disease related gene type.What is more important by the gene mutation site of examination medicaments insensitive, provides foundation for realizing personalized medicine from now on.By finding the gene mutation site of disease susceptibility, can make people's preventing disease early, reach the purpose of the generation of avoiding disease.Therefore, after the human genome sketch had just been completed, gene mutation site became one of main task of the Human Genome Project immediately in the searching human genome.For example, ten big pharmacy giant companies and Wellcome Trust have constituted jointly the SNP cooperative groups in the world, have started the plan of seeking the SNP site in the whole genome scope.China genome center, south and the north (China is big) gene center has also been carried out the screening of the SNP of Chinese nation gene locus.At present, location and certification work have been finished in most human SNP gene polymorphics site (about 3,000,000), and announce on the net.Yet, determine that the gene mutation site in the human genome only is the first step that people studied and utilized gene mutation site, seek gene mutation site and human phenotype, particularly the dependency with human diseases is only people's concern and competition focal point.SNP genotype and functional study thereof have become the integral part of post genome project.
Many methods about sudden change and single nucleotide polymorphism detection are arranged at present, as dna sequencing, restriction enzyme digestion length polymorphism, single strand conformation polymorphism, tetra-sodium order-checking and allele specific oligonucleotide oligonucleotide hybridization etc.Though these technology can be finished the detection to sudden change or nucleotide polymorphisms to a certain extent, at first need target fragment is increased to improve the sensitivity of detection, the site of their detections is quite limited like this, and therefore the information that obtains is incomplete.Develop method simple, cheap, sensitive, that analyze complete genome DNA information reliably for having great importance undoubtedly from full gene level understanding life.
Summary of the invention
Technical problem: the purpose of this invention is to provide a kind of high efficiency full genome fixing method, the form of full genome with fragmentation is separately fixed in the different micropores, preparation complete genome DNA chip, this method has the function that the nucleic acid index amplification of fragmentation is amplified, improved and used the sensitivity that detects, the full genomic information that makes acquisition accurately, reliable.
Technical scheme: the present invention is separately fixed at the form of full genome with fragmentation in the different micropores, constitutes the complete genome DNA chip, and the nucleic acid index amplification of fragmentation is amplified, the full genomic information that makes acquisition accurately, reliable.
High efficiency full genome fixing method of the present invention is:
1.) on a smooth mechanically resistant material, be processed into the garden column type of 5 microns-100 microns of diameters, degree of depth 5-50 micron or square micropore and form microwell plate, and in these micropores stationary phase with oligonucleotide molecules;
2.) genomic fragmentization: with enzyme genomic nucleic acid sequence is cut into the segment that size is the 50-1000 base, and promptly first connexon, second connexon are connected to form and contain connexon genomic fragment nucleotide sequence with a pair of general connexon with these genomic fragment nucleotide sequences under the effect of enzyme;
3.) will contain the dilution of connexon genomic fragment nucleotide sequence thermally denature joins in the hole of microwell plate, and finish hybridization with the fixed oligonucleotide molecules, the multiple that wherein contains connexon genomic fragment nucleotide sequence dilution determines that according to what of micropore each micropore can only be assigned to one at most and contain connexon genomic fragment nucleotide sequence;
4.) amplification reaction solution is joined in the different micropores, increase to containing connexon fragmentation nucleotide sequence then, thereby realized exponentiate amplification that whole genome sequence carries out with the fragmentation form.
Described microwell plate prepares by lithography process, or obtains by excimer laser processing.Stationary phase oligonucleotide molecules together is directly fixing by modifying micropore surface in the micropore, or adopt the gel pre-polymerization zoarium will contain oligonucleotide molecules to be filled in to carry out chemical polymerization in the hole and fix, oligonucleotide molecules should be not complementary with any fragment in the detection genome.The genomic fragment nucleotide sequence connects with a pair of general connexon, is meant that these sequence two ends connect the first general connexon, second connexon respectively, and any segment is all inequality or complementary in it and the genome.Wherein the sequence of 3 ' end, first connexon is complete complementary with the reverse primer sequence oligonucleotides molecule that is fixed in the micropore, 5 ' end, the second connexon sequence with then with reaction system in revocable forward primer complementary fully.Contain connexon genomic fragment nucleotide sequence and determine concentration according to the nucleic acid molecule that can only be assigned to a fragmentation in each micropore on the microwell plate at most.Containing the amplification of connexon genomic fragment nucleotide sequence, is by fixed reverse primer, another on-fixed forward primer in the micropore amplification reaction system genomic fragment nucleotide sequence to be carried out the index amplification; And it is fixed in the micropore effectively, the amplification form is traditional solid-phase amplification, or the solid phase rolling circle amplification.
Mechanically resistant material is glass or silicon chip or gel or plastics or rubber or pottery.
The complete genome DNA micro-array chip that makes up can be used for foranalysis of nucleic acids such as fluorescent hybridization, order-checking.This technology is assigned to genomic full detail in each dna fragmentation, and utilizes PCR method that the genome all information has been realized amplification, makes complete genomic analysis become simple.
Beneficial effect: the present invention compared with prior art has following advantage:
1, great advantage of the present invention is to obtain when having realized full genomic information, by full genome of fragmentation and index these fragments that increase, has saved the template preparation time greatly; Because complete genomic information is amplified with exponential simultaneously, improved the sensitivity that detects simultaneously behind fragmentation.
2, the present invention adopts general connexon to realize being connected of fragmentation nucleic acid and amplification with general amplimer, can save a large amount of detection costs.
3, the purpose of this invention is to provide a kind of complete genome DNA microarray, obtain individual full genomic information by analysis to the complete genome DNA microarray, simultaneously (for example order-checking, hybridization analysis etc.) are realized the obtaining of information in several ways, thereby provide fabulous technical Analysis platform for the high throughput testing of genomic information entirely.
Description of drawings
Fig. 1 is the preparation synoptic diagram of full genome of the present invention and complete genome DNA chip thereof.
Have among the above figure: mechanically resistant material 1, microwell plate 2, oligonucleotide molecules 3, micropore 4,, genomic nucleic acid sequence 5, genomic fragment nucleotide sequence 6, connexon 7, connexon 8, contain connexon genomic fragment nucleotide sequence 9, amplified production 10.
Laser processing A, polymerization be D, solid-phase amplification E under the effect of B, restriction enzyme digestion C, ligase enzyme fixedly.
Embodiment
Genomic dna forms short fragment through suitable digestion with restriction enzyme, the DNA of fragmentation is connected with two general connexons under the effect of enzyme and dilutes the rare solution of poling, make the nucleic acid molecule that can only be assigned to a fragmentation in each micropore at most, and these solution are joined prior processing and in micropore, be fixed with in the microwell plate of corresponding primer.At first the fragmentation nucleic acid molecule sex change in the micropore, make wherein fixed reverse primer annealed combination in a chain and the micropore; Again remaining reaction solution is removed clean; At last the amplification reaction solution that contains another on-fixed forward primer is joined in the micropore, carry out the index amplification in confined conditions, so whole genome sequence with the form of fragmentation respectively by exponentiate be fixed in the different micropores, be built into the genomic dna micro-array chip, it can be used for foranalysis of nucleic acids such as fluorescent hybridization, order-checking.This technology is assigned to genomic full detail in each fragment, and utilizes PCR method that information has been realized amplification, realizes the feasibility that full genome is analyzed on chip.
High efficiency full genome fixing method among the present invention takes following scheme to realize:
1.) on a smooth mechanically resistant material 1, be processed into the garden column type of 5 microns-100 microns of diameters, degree of depth 5-50 micron or square micropore 4 forms microwell plates 2, and in these micropores 4 stationary phase with oligonucleotide molecules 3;
2.) genomic fragmentization: with enzyme genomic nucleic acid sequence 5 is cut into the segment that size is the 50-1000 base, and promptly first connexon 7, second connexon 8 are connected to form and contain connexon genomic fragment nucleotide sequence 9 with a pair of general connexon of these genomic fragment nucleotide sequence 6 usefulness under the effect of enzyme;
3.) will contain the dilution of connexon genomic fragment nucleotide sequence 9 thermally denatures joins in the hole of microwell plate 2, and finish hybridization with fixed oligonucleotide molecules 3, the multiple that wherein contains connexon genomic fragment nucleotide sequence 9 dilution determines that according to what of micropore 4 each micropore 4 can only be assigned to one at most and contain connexon genomic fragment nucleotide sequence 9;
4.) amplification reaction solution is joined in the different micropore 4, increase to containing connexon fragmentation nucleotide sequence (9) then, thereby realized exponentiate amplification that whole genome sequence carries out with the fragmentation form.
Be processed into the microwell plate of 5 microns-100 microns of diameters, degree of depth 5-50 micron on a smooth mechanically resistant material, it can prepare by lithography process, also can obtain by the laser calcination.And in these micropores stationary phase with oligonucleotide molecules, can be directly fixing by modifying micropore surface, also can adopt the gel pre-polymerization zoarium that will contain oligonucleotide molecules to be filled in to carry out chemical polymerization in the hole and fix.Oligonucleotide molecules should be not complementary with any fragment in the detection genome.
Earlier genome is cut into the segment that size is the 50-1000 base with restriction enzyme, and under the effect of ligase enzyme, these fragmentation nucleotide sequences are connected with a pair of general connexon, institute's fixed reverse primer sequence is complementary fully in one of them connexon and the micropore plate hole; Another connexon is complete complementation with carrying out PCR another on-fixed forward primer of when reaction.
The dilution of the genomic DNA fragment of fragmentation is joined in the micropore, and finish hybridization with immobilized primer, wherein the dilution of fragmentation genome is decided on what of micropore, and each micropore can only be assigned to the dna fragmentation of a fragmentation at most.Assurance does not have two dna fragmentations to enter in the same micropore, therefore allows the dna fragmentation that does not have fragmentation in some micropores.To add the dna fragmentation sex change in the micropore then, make fixed forward primer hybridization in one bar chain and the micropore, then remaining reaction liquid be removed.
The amplification reaction solution that will comprise another on-fixed reverse primer joins in the micropore, immobilization forward primer in micropore, another on-fixed reverse primer and fragmentation nucleic acid molecule with uniqueness constitute little reaction system like this, after they carry out the index amplification in confined conditions, then whole genome sequence is fixed on respectively in the different micropores with the form of fragmentation, thereby has been built into the complete genome DNA micro-array chip.
The invention will be further described below with reference to accompanying drawing.
Among Fig. 1, a smooth mechanically resistant material 1, can be plastics, glass etc., become the microwell plate 2 of 5 microns-100 microns of diameters, degree of depth 5-50 micron by photoetching or excimer laser processing A, then with the primer 3 modified by directly or chemical polymerization fixedly B in these micropores 4, make micropore surface be fixed with identical oligonucleotide molecules 3; On the other hand, the genomic dna that places the Eppendorff pipe is by using restriction enzyme digestion C, genome 5 fragments are turned to the genomic fragment nucleotide sequence 6 of big or small 50-1000 base, these fragmentation nucleotide sequences D under the effect of ligase enzyme is connected with 8 with a pair of general connexon 7, and wherein fixed oligonucleotide molecules 3 sequences are complete complementations in connexon 7 and the micropore plate hole; The amplimer that adds in connexon 8 and the PCR reaction solution is complete complementation.To contain the full genomic fragment nucleotide sequence 9 of connexon joins in the microwell plate of immobilized oligonucleotide molecule 3, make and to be assigned to one in each micropore at most and to contain the full genomic fragment nucleotide sequence 9 of connexon, by sex change, make fixed oligonucleotide molecules 3 hybridization in one bar chain and the micropore.After remaining reaction liquid was removed, the amplification reaction solution that will comprise amplimer joined in the micropore, carries out solid-phase amplification E in confined conditions and obtains amplified production 10.Whole genome sequence is increased afterwards and is fixed by exponentiate in different micropores with the form of fragmentation like this, thereby is built into the genomic dna micro-array chip.
Full genome of example 1 yeast and complete genome DNA chip production method thereof
With reference to accompanying drawing 1, on a smooth sheet glass by lithography process become that diameter is 50 little, the microwell plate of 50 microns of the degree of depth, with dichlorodimethylsilane hydrophobic treatment is carried out on the microwell plate surface then.Micropore through washing, dry back with 5% 3-TSL 8330 acetone modify 1 hour, 5% glutaraldehyde processing after 2 hours with the oligonucleotide solution reaction of 5 ' the terminal modified amine groups, oligonucleoside is fixed in the micropore.On the other hand, the yeast genes group is cut into the segment that size is about 500 bases with enzyme, and under the effect of ligase enzyme, these fragmentation nucleotide sequences are connected with a pair of general connexon, fixed oligonucleotide molecules sequence is complementary fully in one of them connexon and the micropore plate hole, and another connexon is then complementary fully with amplimer.
The fragmentation nucleotide sequence dilution that will contain connecting arm joins in the microwell plate, and the multiple of dilution is the nucleic acid molecule that can only be assigned to a fragmentation on the microwell plate in each micropore at most, finishes the hybridization with fixed oligonucleotide molecules sequence then.
The amplification reaction solution that will comprise amplimer joins that the nucleotide sequence to fragmentation increases in the micropore, so the fragmentation form ground of whole genome sequence with the exponentiate amplification is separately fixed in the different microwell plates formation complete genome DNA chip.Utilize this complete genome DNA chip (for example order-checking, hybridization analysis etc.) realization obtaining in several ways to the full genomic information of yeast.
Full genome of example 2 people and complete genome DNA chip production method thereof
With reference to accompanying drawing 1, on a smooth sheet glass, become 50 microns of diameters, dark 50 microns microwell plate by lithography process, with dichlorodimethylsilane hydrophobic treatment is carried out on the microwell plate surface then.With the oligonucleotide of 5 ' the terminal modified acrylamide group be filled in the micropore after acrylamide monomer, ammonium persulphate etc. mixes, and allow the water in the prepolymer volatilize naturally, when evaporating into prepolymer, water only accounts for 2/3rds to three/for the moment of micropore, sheet glass is placed under the vacuum, and short its polymerization reaction take place forms gel and is fixed in the micropore under the atmosphere of initiator vaporization.
On the one hand, the people's gene group is cut into the segment that size is the 50-1000 base with enzyme, and under the effect of ligase enzyme, these fragmentation nucleotide sequences are connected with a pair of general connexon.The immobilized oligonucleotide molecular sequences is complete complementation in one of them connexon and the micropore plate hole; Another general connexon is then complementary fully with revocable amplimer.
On the other hand, the fragmentation nucleotide sequence dilution that contains connexon is joined in the microwell plate, the multiple of dilution is decided according to being assigned to the nucleic acid molecule of a fragmentation at most in each micropore on the microwell plate.Then itself and immobilization primer are hybridized.At last the amplification reaction solution that comprises forward primer is joined that the nucleic acid molecule to fragmentation increases in the micropore.So just the form of complete genomic fragment with the exponentiate amplification is separately fixed in the different microwell plates, constitutes the complete genome DNA chip.Utilize (for example order-checking, the hybridization analysis etc.) realization in several ways of this complete genome DNA chip to the analysis of the full genomic information of people.
Claims (7)
1, a kind of high efficiency full genome fixing method is characterized in that this method is:
1.) on a smooth mechanically resistant material (1), be processed into the garden column type of 5 microns-100 microns of diameters, degree of depth 5-50 micron or square micropore (4) and form microwell plate (2), and in these micropores (4) the same oligonucleotide molecules (3) of stationary phase;
2.) genomic fragmentization: with enzyme genomic nucleic acid sequence (5) is cut into size and be the segment of 50-1000 base, and promptly first connexon (7), second connexon (8) are connected to form and contain connexon genomic fragment nucleotide sequence (9) with a pair of general connexon with these genomic fragment nucleotide sequences (6) under the effect of enzyme;
3.) will contain the dilution of connexon genomic fragment nucleotide sequence (9) thermally denature joins in the hole of microwell plate (2), and finish hybridization with fixed oligonucleotide molecules (3), the multiple that wherein contains connexon genomic fragment nucleotide sequence (9) dilution determines that according to what of micropore (4) each micropore (4) can only be assigned to one at most and contain connexon genomic fragment nucleotide sequence (9);
4.) amplification reaction solution is joined in the different micropore (4), increase to containing connexon fragmentation nucleotide sequence (9) then, thereby realized exponentiate amplification that whole genome sequence carries out with the fragmentation form.
2, high efficiency full genome fixing method according to claim 1 is characterized in that described microwell plate (2), prepares by lithography process, or obtains by excimer laser processing.
3, high efficiency full genome fixing method according to claim 1, it is characterized in that stationary phase oligonucleotide molecules (3) together is directly fixing by modifying micropore surface in the micropore (4), or adopt the gel pre-polymerization zoarium will contain oligonucleotide molecules to be filled in to carry out chemical polymerization in the hole and fix, oligonucleotide molecules should be not complementary with any fragment in the detection genome.
4, high efficiency full genome fixing method according to claim 1, it is characterized in that genomic fragment nucleotide sequence (6) connects with a pair of general connexon, be meant that these sequence two ends connect general first connexon (7), second connexon (8) respectively, any segment is all inequality or complementary in it and the genome.Wherein the sequence of 3 ' end first connexon (7) is complete complementary with the reverse primer sequence oligonucleotides molecule (3) that is fixed in the micropore, 5 ' end second connexon (8) sequence with then with reaction system in revocable forward primer complementary fully.
5, high efficiency full genome fixing method according to claim 1 is characterized in that containing connexon genomic fragment nucleotide sequence (9) and determines concentration according to the nucleic acid molecule that can only be assigned to a fragmentation in each micropore on the microwell plate at most.
6, high efficiency full genome fixing method according to claim 1, it is characterized in that containing the amplification of connexon genomic fragment nucleotide sequence (9), is by fixed reverse primer, another on-fixed forward primer in the micropore amplification reaction system genomic fragment nucleotide sequence to be carried out the index amplification; And it is fixed in the micropore effectively, the amplification form is traditional solid-phase amplification, or the solid phase rolling circle amplification.
7,, it is characterized in that mechanically resistant material (1) is glass or silicon chip or gel or plastics or rubber or pottery according to the high efficiency full genome fixing method of claim 1 indication.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100386445C (en) * | 2007-04-17 | 2008-05-07 | 中南大学 | Genome chip for analyzing microbial community structure in acidic environment |
| CN107557269A (en) * | 2013-02-26 | 2018-01-09 | 伊鲁米那股份有限公司 | The surface of gel medelling |
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2006
- 2006-10-20 CN CN 200610096841 patent/CN1944675A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100386445C (en) * | 2007-04-17 | 2008-05-07 | 中南大学 | Genome chip for analyzing microbial community structure in acidic environment |
| CN107557269A (en) * | 2013-02-26 | 2018-01-09 | 伊鲁米那股份有限公司 | The surface of gel medelling |
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