CN110229903A - Molecular marker of the PODN as Diagnosis of Thyroid Carcinoma - Google Patents
Molecular marker of the PODN as Diagnosis of Thyroid Carcinoma Download PDFInfo
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- CN110229903A CN110229903A CN201910554518.2A CN201910554518A CN110229903A CN 110229903 A CN110229903 A CN 110229903A CN 201910554518 A CN201910554518 A CN 201910554518A CN 110229903 A CN110229903 A CN 110229903A
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- podn
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- albumen
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Classifications
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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
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Abstract
The invention discloses the molecular marker-PODN early diagnosed for thyroid cancer.Research of the present invention has shown that compared with cancer beside organism, the mRNA expression of PODN gene is remarkably decreased in thyroid cancer patients.According to the existing correlation between PODN gene and thyroid cancer, the kit of Diagnosis of Thyroid Carcinoma can be prepared, which can clinically be widely applied.
Description
Technical field
The present invention relates to diagnosis of thyroid cancer fields, more particularly it relates to which PODN gene is as thyroid cancer
Diagnostic tool.
Background technique
Thyroid cancer is common endocrine tumors, and having 5%-10% in the thyroid nodule of clinical discovery is thyroid gland
Cancer.Differentiated thyroid carcinoma accounts for about the 90% of all thyroid cancers, including papillary adenocarcinoma and follicular adenocarcinoma, other are more rare
Have anaplastic thyroid carcinoma and medullary carcinoma of thyroid gland etc..Investigation display China's thyroid cancer incidence increases year by year.First shape
There are the malaise symptoms such as compressing except gland benign protuberance, it may not be necessary to it performs the operation, and thyroid cancer then needs excision of performing the operation as early as possible.Therefore, such as
What accurately makes diagnosis to thyroid nodule early stage, it appears particularly important.Fine-needle aspiration of thyroid nodules cytolgical examination (FNAB)
It is presently believed to be the most effective method for identifying tubercle property, but this method is also only the thyroid nodule of 70-85%
Offer is clarified a diagnosis.And this method is by the restriction for puncturing doctor and pathological diagnosis doctor's clinical experience, some tubercle properties
It is difficult to identify from cytology.Therefore, a kind of high sensitivity, high specificity are found, the few diagnostic method of restraining factors is urgently
It solves the problems, such as.
Summary of the invention
The purpose of the present invention is to provide one kind by detection PODN gene or protein expression difference come Diagnosis of Thyroid Carcinoma
Method.
To achieve the goals above, present invention employs following technical solutions:
The present invention provides the products of detection PODN gene or PODN albumen to prepare the use in diagnosis of thyroid cancer tool
On the way.
Further, the product of the detection PODN gene or PODN albumen includes the table for detecting PODN gene or PODN albumen
Up to horizontal product.The product includes the nucleic acid that can combine PODN gene or the substance (example that can combine PODN albumen
Such as antibody).The nucleic acid is able to detect the expression of PODN gene；The substance is able to detect the expression water of PODN albumen
It is flat.
The product of detection PODN gene of the invention can play its function based on the known method of nucleic acid molecules is used: such as
PCR, such as Southern hybridization, Northern hybridization, dot blot, fluorescence in situ hybridization (FISH), DNA microarray, ASO method, height
Flux microarray dataset etc..It can qualitatively, quantitatively or semi-quantitatively implement analysis using the product.
Include that nucleic acid in the said goods can be obtained by chemical synthesis, or by containing from biomaterial preparation
It is expected that the gene of nucleic acid, then using primer amplification designed for amplification expectation nucleic acid, it is obtained.
Further, the PCR method is known method, for example, ARMS (Amplification Refractory
Abruptly-changing system is not answered in Mutation System, amplification) method, RT-PCR (reverse transcriptase-PCR) method, nesting PCR method etc..Amplification
Nucleic acid can be by using dot blotting hybridization method, Surface Plasmon Resonance (SPR method), PCR-RFLP method, original position RT-PCR
Method, PCR-SSO (sequence specific oligonucleotide) method, PCR-SSP method, AMPFLP (amplifiable fragment length polymorphism) method,
MVR-PCR method and PCR-SSCP (single-strand conformation polymorphism) method detect.
Nucleic acid recited above includes the primer for expanding PODN gene, and the primer for including in product can be by passing through chemistry
Synthesis to prepare, by using those skilled in the art will know that method be suitably designed with reference to Given information, and passing through
Synthesis is learned to prepare.
In specific embodiments of the present invention, the nucleic acid is amplimer used in QPCR experiment, the primer
Sequence such as SEQ ID NO.1 (positive sequence) and SEQ ID NO.2 (reverse sequence) shown in.
Nucleic acid recited above may also include probe, and the probe can be prepared by chemical synthesis, by using this
The method that field technical staff knows appropriately is designed with reference to Given information, and is prepared by chemical synthesis, or can lead to
The gene for containing desired nucleic acid sequence from biomaterial preparation is crossed, and is expanded using the primer designed for amplification expectation nucleic acid sequence
Increase it to prepare.
The product of detection PODN albumen of the invention can play its function based on the known method of antibody is used: for example,
It may include ELISA, radioimmunoassay, immunohistochemical method, western blot etc..
The product of detection PODN albumen of the invention includes the antibody or its segment for specifically binding PODN albumen.It can make
With the antibody or its segment of any structure and size, immunoglobulin class, origin etc., as long as it combines target protein.This
The antibody or its segment for including in the testing product of invention can be monoclonal or polyclonal.Antibody fragment refers to reservation antibody
Peptide to the active antibody of the combination of antigen a part of (Partial Fragment) or containing antibody a part.Antibody fragment may include F
(ab′)2, Fab ', Fab, scFv (scFv), the Fv (dsFv) of disulphide bonding or its polymer, the area dimerization V it is (dual anti-
Body) or peptide containing CDR.The product of detection PODN albumen of the invention may include encoding antibody or Encoding Antibody Fragment
The isolated nucleic acid of amino acid sequence, the carrier comprising the nucleic acid, and carry the cell of the carrier.
Antibody can be obtained by the way that well known to a person skilled in the art methods.For example, preparation retains target all or in part
The mammalian cell expression vector of their polynucleotides of the polypeptide or integration coding of protein is as antigen.Exempted from using antigen
After epidemic disease animal, from the immune animal adaptive immune cell of process and myeloma cell is merged to obtain hybridoma.Then from hybridization
Tumor culture collects antibody.It can finally be implemented by using antibody of the PODN albumen for being used as antigen or part thereof to acquisition
Antigentic specificity purifies to obtain the monoclonal antibody for PODN albumen.Polyclonal antibody can be prepared as follows: with it is above
Identical antigen-immunized animal collects blood sample from by immune animal, serum is isolated from blood, then using upper
It states antigen and antigentic specificity purifying is implemented to serum.It can be by the antibody that is obtained with enzymatic treatment or by using the antibody of acquisition
Sequence information obtain antibody fragment.
The combination of marker and antibody or its segment can be implemented by method as commonly known in the art.For example, can
With following fluorescent marker protein or peptide: clean protein or peptide with phosphate buffer, addition DMSO, buffer, etc. standards
Standby dyestuff, then mixed solution, then at being placed at room temperature for 10 minutes.In addition, the labelling kit of commercialization can be used in label, it is all
Such as biotin labeling reagent box, such as biotin labeling reagent box-NH2, biotin labeling reagent box-SH
(DojindoLaboratories)；Alkali phosphatase enzyme mark kit such as alkali phosphatase enzyme mark kit-NH2, alkaline phosphorus
Sour enzyme labelling kit-SH (Dojindo Laboratories)；Peroxidase labelling kit such as peroxidase mark
Remember kit-NH2, peroxidase labelling kit-NH2 (Dojindo Laboratories)；Phycobniliprotein labelled reagent
Box such as phycobniliprotein labelling kit-NH2, phycobniliprotein labelling kit-SH, B- phycoerythrin labelling kit-NH2,
B- phycoerythrin labelling kit-SH, R-PE labelling kit-NH2, R-PE labelling kit SH
(DojindoLaboratories)；Fluorescent labeling reagent box such as fluorescein labelling kit-NH2, HiLyte Fluor (TM)
555 labelling kit-NH2,647 labelling kit-NH2 of HiLyte Fluor (TM) (Dojindo Laboratories)；And
DyLight 547 and DyLight647 (Techno Chemical Corp.), Zenon (TM), Alexa Fluor (TM) antibody
Labelling kit, Qdot (TM) antibody labeling kit (Invitrogen Corporation) and EZ- marker protein mark
Remember kit (Funakoshi Corporation).For correct labeling, suitable instrument can be used to detect by label
Antibody or its segment.
Further, the product of the detection PODN gene or PODN albumen can be detection PODN gene or PODN albumen
Reagent is also possible to include kit, chip, test paper of the reagent etc., is also possible to measure using the high pass of the reagent
Sequence platform.
The expression of the PODN gene or PODN albumen in subject's sample is detected using mentioned-above testing product,
It is compared with normal people, the expression of PODN gene or PODN albumen in subject's sample reduces, then diagnosing the subject is
Thyroid cancer patients diagnose risk height of the subject with thyroid cancer.
As the sample according to testing product of the invention, the tissue sample for example obtained from biopsy subject can be used
Or fluid.Sample is not particularly limited, as long as it is suitable for measurement of the invention；For example, it may include tissue, blood, blood plasma,
Serum, lymph, urine, serous cavity liquid, spinal fluid, synovia, aqueous humor, tear, saliva or its fraction or treated material
Material.
In specific embodiments of the present invention, tissue of the sample from subject.
The present invention also provides a kind of tool of Diagnosis of Thyroid Carcinoma, the tool is able to detect PODN in subject's sample
The expression of gene or PODN albumen.The tool includes that can combine the nucleic acid of PODN gene or can combine PODN egg
White substance (such as antibody).The nucleic acid is able to detect the expression of PODN gene；The substance is able to detect PODN egg
White expression.
Further, the property of the nucleic acid and the substance is the same as noted earlier.
Further, the tool of the Diagnosis of Thyroid Carcinoma includes but is not limited to that chip, kit, test paper or high pass measure
Sequence platform；High-flux sequence platform is a kind of tool of special Diagnosis of Thyroid Carcinoma, with the development of high throughput sequencing technologies,
Very easily work will be become to the building of the gene expression profile of a people.By the base for comparing Disease and normal population
Because of express spectra, the exception for being easy to analyze which gene is related to disease.Therefore, PODN gene is known in high-flux sequence
The abnormal purposes for also belonging to PODN gene related to thyroid cancer, equally within protection scope of the present invention.
The number for the amino acid that anti-PODN antibody used in testing product of the invention, diagnostic tool or its segment are identified
Mesh is not particularly limited, as long as antibody can combine PODN.When antibody is as therapeutic agent, preferably it can know
Amino acid not as much as possible, as long as it can inhibit PODN function.The number of antibody or the amino acid of its segment identification is at least
One, more preferably at least three.The immunoglobulin class of antibody is unrestricted, can be IgG, IgM, IgA, IgE, IgD or
IgY。
Other properties of anti-PODN antibody used in testing product of the invention, diagnostic tool are the same as noted earlier.
Further, the tissue sample or fluid for example obtained from biopsy subject can be used in subject's sample.Sample
Originally it is not particularly limited, as long as it is suitable for measurement of the invention；For example, it may include tissue, blood, blood plasma, serum, lymph
Liquid, urine, serous cavity liquid, spinal fluid, synovia, aqueous humor, tear, saliva or its fraction or treated material.In the present invention
Specific embodiment in, tissue of the sample from subject.
The present invention also provides a kind of methods of Diagnosis of Thyroid Carcinoma, and described method includes following steps:
(1) sample of thyroid cancer subject is obtained；
(2) expression of PODN gene or albumen in Samples subjects is detected；
(3) it associates whether by the expression of the PODN gene or albumen that measure with the illness of subject.
(4) compared with normal control, the expression of PODN gene or albumen is reduced, then the subject is diagnosed as first shape
It is high that gland cancer or the subject are diagnosed as the risk with thyroid cancer in the future.
In the context of the present invention, " Diagnosis of Thyroid Carcinoma " both includes judging whether subject has suffered from thyroid gland
Cancer also includes the risk that judges subject and whether there is with thyroid cancer.
Information of " the PODN gene " of the invention on NCBI are as follows: Chromosome 1, NC_000001.11
(53062052..53085502)。
The advantages of the present invention:
The present invention for the early diagnosis of thyroid cancer disease, Index for diagnosis and early intervention treatment provide important reference according to
According to, there is biggish actual clinical value, can be used for filtering out the people at highest risk and recurrence crowd of thyroid cancer disease, to
Intervened early and treated, reduces the unnecessary treatment of non-high-risk recurrence patient and health care costs.
Detailed description of the invention
Fig. 1, which is shown, utilizes differential expression of the PODN gene in cancerous tissue and cancer beside organism in QPCR detection tissue.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this
It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
The differential expression of PODN gene in 1 cancer beside organism of embodiment and human thyroid carcinoma
1, research object:
Collect the patient that 30 are diagnosed as thyroid cancer through hospital human thyroid carcinoma and corresponding cancer beside organism.
2, the differential expression of PODN gene is detected on transcriptional level
The extraction of 2.1 tissue RNA
1) in the clear area of less RNase interference, in vitro tissue sample about 20mg is weighed using the mortar containing appropriate liquid nitrogen,
It is ground to pestle powdered；
2) sample is transferred in the centrifuge tube of a 2mL without RNA enzyme；
3) 300 μ l Lysis solution are added, is placed in homogenizer, is fully ground 1-5min；
4) 12000g, is centrifuged 10min by 4 DEG C, shifts supernatant into the centrifuge tube of new 1.5mL；
5) 600 μ l RNase-Free Water are added, are mixed with vortex device；
6) 20 μ l Proteinase Ks are added, in 55 DEG C of warm bath 15min, is constantly vortexed and mixes；
7) 14000g, room temperature are centrifuged 1min, make pellet cell debris in centrifugation bottom of the tube, supernatant is taken to be transferred to other one
In a centrifuge tube without RNA enzyme 1.5mL；
8) 95% ethyl alcohol of 450 μ l is added, is vortexed and mixes；
RNA absorption:
9) 650 lysates of the μ l containing ethyl alcohol are taken to be added in centrifugal column, 14000g is centrifuged 1min；
10) lower layer is abandoned, resets collecting pipe on column；
11) according to the capacity of lysate, repeat 9)~10) step；
12) 400 μ l Wash solution, 14000g are added and are centrifuged 2min；
13) lower layer is abandoned, column is placed on a new collecting pipe；
DNase processing:
14) 100 μ l Enzyme Incubation Buffer are added and 15 μ l DNase I, 14000g are centrifuged 1min；
15) solution in collecting pipe is moved into column again；
16) it is placed at room temperature for 15min；
RNA washing:
17) 400 μ l Wash solution, 14000g are added and are centrifuged 1min, abandons lower layer, resets collecting pipe on column；
18) 400 μ l Wash solution, 14000g are added and are centrifuged 2min, abandon collecting pipe；
RNA elution:
19) pillar is put into 1.7mL Elution pipe；
20) 30 μ l Elution Buffer are added；
21) 200g is centrifuged 2min, makes solution sufficiently in conjunction with column, and then 14000g is centrifuged 1min.
2.2 reverse transcription
The reverse transcription of RNA is carried out using the Reverse Transcriptase kit of TAKARA company.
2.3QPCR
(1) design of primers
QPCR amplimer is designed according to the coded sequence of PODN gene and GAPDH gene in Genbank, by the raw work in Shanghai
The synthesis of biotechnology Services Co., Ltd.Specific primer sequence is as follows:
PODN gene:
Forward primer is 5 '-AGAACAACAAGATTAGTG-3 ' (SEQ ID NO.1)；
Reverse primer is 5 '-GTTAAACCTGAGAAAGAT-3 ' (SEQ ID NO.2),
GAPDH gene:
Forward primer is 5 '-TTTAACTCTGGTAAAGTGGATAT-3 ' (SEQ ID NO.3)；
Reverse primer is 5 '-GGTGGAATCATATTGGAACA-3 ' (SEQ ID NO.4).
(2) PCR reaction system is prepared according to table 1:
Wherein, SYBR Green polymerase chain reaction system is purchased from Invitrogen company.
Table 1PCR reaction system
| Reagent | Volume |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| SYBR Green polymerase chain reaction system | 12.5μl |
| Template | 2μl |
| Deionized water | Supply 25 μ l |
(3) PCR reaction condition: 95 DEG C of 5min, (95 DEG C of 20s, 57 DEG C of 40s) * 40 circulations.Using SYBR Green as glimmering
Signal object carries out PCR reaction on Light Cycler fluorescence quantitative PCR instrument, is determined by melt curve analysis analysis and electrophoresis
Purpose band, Δ Δ CT method carry out relative quantification.
2.4 statistical method
Experiment is completed according to being repeated 3 times, and result data is indicated in a manner of mean+SD,
Using SPSS13.0 statistical software come for statistical analysis, the difference between different groups is examined using t, it is believed that as P < 0.05
With statistical significance.
2.5 result
The results show that there is the mRNA of PODN gene in 29 patient's cancerous tissues to be substantially less than cancer in 30 thyroid cancer patients
Side tissue, as shown in Figure 1, difference has statistical significance (P < 0.05) to statistical result.
The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this
For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention
And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.
Sequence table
<110>Taizhou municipal hospital
<120>molecular marker of the PODN as Diagnosis of Thyroid Carcinoma
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
agaacaacaa gattagtg 18
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gttaaacctg agaaagat 18
<210> 3
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tttaactctg gtaaagtgga tat 23
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<400> 4
ggtggaatca tattggaaca 20
Claims (10)
1. the product of detection PODN gene or PODN albumen is preparing the application in diagnosis of thyroid cancer tool.
2. application according to claim 1, which is characterized in that the detection PODN gene or the product of PODN albumen include
Detect the product of the expression of PODN gene or PODN albumen.
3. application according to claim 2, which is characterized in that utilize the PODN base in the product testing subject sample
The expression of cause or PODN albumen, is compared with normal people, the expression water of PODN gene or PODN albumen in subject's sample
It is flat to lower, then the subject is diagnosed as thyroid cancer patients or diagnoses risk height of the subject with thyroid cancer.
4. application according to claim 3, which is characterized in that the source of subject's sample is tissue.
5. application described in any one of -4 according to claim 1, which is characterized in that the product includes that can combine PODN base
The nucleic acid of cause can be in conjunction with the substance of PODN albumen；The nucleic acid is able to detect the expression of PODN gene；The object
Matter is able to detect the expression of PODN albumen.
6. application according to claim 5, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
Increase the primer of PODN gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
7. a kind of tool of Diagnosis of Thyroid Carcinoma, which is characterized in that the tool includes being able to detect in subject's sample
The tool of the expression of PODN gene or PODN albumen.
8. tool according to claim 7, which is characterized in that the tool include can in conjunction with PODN gene nucleic acid or
Person can be in conjunction with the substance of PODN albumen；The nucleic acid is able to detect the expression of PODN gene；The substance is able to detect
The expression of PODN albumen.
9. tool according to claim 8, which is characterized in that the nucleic acid is specifically expanded used in real-time quantitative PCR
Increase the primer of PODN gene as shown in SEQ ID NO.1 and SEQ ID NO.2.
10. the tool according to any one of claim 7-9, which is characterized in that the source of subject's sample is group
It knits.
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