CN103233064B - Primer probe system, method and kit for detecting epidermal growth factor receptor 19, 21 exon mutations - Google Patents

Abstract

The present invention relates to a primer probe system, a method and a kit for detecting epidermal growth factor receptor exon 19 and 21 mutations. According to the method, an asymmetric PCR technology, a nucleic acid isothermal amplification technology and a fluorescence detection technology are organically integrated, wherein the asymmetric PCR amplification technology and special primers are adopted to obtain a lot of single-stranded DNA so as to overcome disadvantages of low efficiency linear amplification of 3WJ (an isothermal digestion nucleic acid detection technology), and specificity and simplicity of the 3WJ technology are adopted, such that a reaction temperature can be controlled at about 30 DEG C, enzyme activity reduction can not be generated, expensive and precise temperature control equipment is not required during a detection process, and discrimination can be achieved only through the simple fluorescence detection instrument with the special probe design. According to the present invention, advantages of rapidness, simpleness, specificity, sensitivity, and the like are provided.

Description

Detection of epidermal growth factor receptor 19 , twenty one Primer probe system, method and kit for exon mutation

technical field

The invention relates to the technical field of bioengineering, in particular to a primer probe system, method and kit for detecting mutations in exons 19 and 21 of epidermal growth factor receptors.

Background technique

Lung cancer, especially non-small cell lung cancer, is one of the most malignant tumors in the world. Although traditional chemotherapy has been continuously improved, the prognosis of advanced patients is relatively poor. With the improvement of molecular technology, targeted drugs targeting EGFR (epidermal growth factor receptor) - tyrosine kinase inhibitors (TKI) are becoming more and more prominent in the treatment of NSCLC, among which gefitinib and erythridine Rotinib has been widely used in the treatment of NSCLC. With the accumulation of clinical data, in the treatment of NSCLC, some people respond to TKI treatment. By analyzing the EGFR gene of these TKI-treated patients, it was found that almost all of them had EGFR gene mutations. A large number of studies have shown that EGFR gene mutations in lung cancer patients mainly occur in exons 18-21, and more than 90% of the mutations are 15 base deletions in exon 19 and L858R mutations in exon 21. These mutations are related to the efficacy of TKI, and the possible reason is that these mutations change the protein structure of EGFR ATP-binding region, making this region easy to bind with TKI, thus improving the efficacy of TKI therapy. Therefore, it is very necessary to detect EGFR gene mutations in patients before clinical use of TKI therapy. Because in our country, EGFR gene mutations are mainly concentrated in exon 19 and exon 21. Therefore, it is very important to quickly and accurately detect EGFR mutations to guide clinical medication and improve the process of personalized medicine in my country.

Since the advent of PCR technology, the technique has been widely used in molecular diagnosis. Mutation detection technology based on PCR technology has been developed rapidly: PCR-RFLP, allele-sespecific PCR, TaqMan probe, ARMS, PNA-LNA clamp fluorescent PCR, Cycleave probe PCR, Nanofluidic Digital PCR Arrays, MassArray and DNA chips and more. PCR-RFLP is an early experimental development to detect SNP typing technology, which relies on the skillful selection of restriction endonucleases to cut out short fragment length polymorphisms for identifying mutation sites, but this method requires electrophoresis separation after enzyme digestion products, which would severely limit the throughput of the reaction and the automation of the operation. ARMS technology is roughly the same as allele-sespecific PCR. It uses DNA polymerase to lack 3'~5' exo-cutting activity, and the extension speed of the mismatched primer at the 3' end is lower than that of the normal primer. When the number of mismatches reaches a certain degree of stringency , the 3' end cannot be extended. If there is a band in PCR, it means that there is a corresponding mutation. ARMS technology has high sensitivity and specificity, and has been successfully applied in clinical detection of SNP.

TaqMan probes are oligonucleotide probes with a fluorescent reporter group attached to the 3' end of the probe and a fluorescent quencher group attached to the 5' end. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group. When PCR is amplified, the 5' exonuclease activity of Taq enzyme can bind to the quencher group of the probe on the target DNA chain After cutting off, the fluorescent reporting group and the fluorescent quenching group of the probe are separated, so that the fluorescent monitoring system can receive the fluorescent signal. By using different fluorescent reporter groups, different enzyme-digested allele-specific probes can be detected simultaneously in the same PCR. This 5' exonuclease assay has been successfully used to distinguish alleles that differ by only one base.

Cycleave probe PCR, the technical principle is similar to the Taqman probe, except that the probe contains an rNTP base based on SNP complementation. When there is a mutation, the rNTP is paired with the mutation, and RNaseH recognizes and cuts the rNTP, so that the fluorescent reporter group of the probe and the fluorescence quencher The killing group is separated and fluorescence is produced.

PNA-LNA-clamp technology, using NestPCR technology and PNA-LNA-clamp probe, PNA-LNA-clamp probe specifically blocks wild SNP, prevents wild template amplification and achieves the purpose of amplifying mutations. While this technique is sensitive, it is susceptible to contamination and limited in probe design by undergoing two PCRs.

Although the above-mentioned SNP detection techniques have high sensitivity and specificity, these techniques require sophisticated and expensive temperature-controlled temperature and fluorescence detection equipment. Therefore, it is of great significance to develop an economical and practical SNP detection method. At present, the gold standard of SNP mutation detection technology is "DNA sequencing", but the sensitivity of DNA sequencing is relatively low, and <20% nucleic acid mutations are difficult to distinguish, and the cycle is relatively long.

Nucleic acid constant temperature amplification technology such as constant temperature enzyme digestion detection technology (3WJ, three way junction) invader technology is gradually being applied to mutation detection. 3WJ was proposed by Amicarelli G et al. Its technical principle is as follows: two probes PA and PB form the core component of 3WJ, PB is used as an anchor probe to bind to the template, PA is used as a resolution probe to recognize SNP, and the 3' end has BHQ Quenching group, with a fluorescent group FAM upstream of the 5' end of the enzyme cutting site; there are 6~8 complementary bases between PA and PB, which contains a restriction endonuclease cutting site (5'-GTAC-3 '), when the SNP to be detected exists, PA and PB form a stable "Y"-shaped structure with the target sequence, restriction endonucleases such as CviQ1 recognize and cut off the quencher group-BHQ, and fluorescence is generated.

Although JP and 3WJ technologies can distinguish SNP polymorphisms well, their linear amplification efficiency and sensitivity are far below the ability to detect DNA in clinical samples, and more importantly, they can only detect single-stranded DNA.

Contents of the invention

The technical problem to be solved by the present invention is to provide a highly specific and sensitive primer probe system for detecting mutations in exons 19 and 21 of the epidermal growth factor receptor, and combine asymmetric PCR with nucleic acid constant temperature amplification and fluorescence detection A fast, simple, specific, sensitive, stable, and low-cost method for detecting mutations in exons 19 and 21 of the epidermal growth factor receptor was designed by combining technologies, and a simple and practical method for detecting epidermal growth factor receptor A kit for mutations in exons 19 and 21 of growth factor receptors.

In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:

A primer-probe system for detecting mutations in exons 19 and 21 of the epidermal growth factor receptor was designed, including asymmetric PCR primers for amplifying the E746-A750del mutation in exon 19 of EGFR and the L858R mutation in exon 21 of EGFR as follows: And the 3WJ probe matching its primer is as follows:

Among them, FAM (fluorescence group) is marked at the 11th position of probe A, and DABCY is marked at the 3' end; FAM is marked at the 7th position of probe A', and DABCY is marked at the 3' end.

A method for detecting epidermal growth factor receptor exon 19, 21 mutations, comprising the steps of:

(1) Process the samples to be tested according to conventional methods;

(2) PCR amplification: use the above-mentioned upstream primer A and downstream primer B for detecting 19 DEL to form the following asymmetric PCR system to amplify EGFR19 exon E746～A750del:

5×buffer 10 microliters

2.5 µl of 25 mM MgCl ₂

10 mM dNTP 1 microliter

10 μM upstream primer A 3 microliters

10 μM downstream primer B 0.2 μl

500 units of Taq DNA Polymerase 0.3 µl

ddH ₂ O 33 µl

total 50 microliters

The PCR amplification conditions are as follows:

Replace the upper and lower primers in the above-mentioned asymmetric PCR system with the above-mentioned upstream primer A' and downstream primer B' corresponding to the detection of L858R, and the formed asymmetric PCR system amplifies exon 21 according to the same PCR amplification conditions L858R;

(3) 3WJ detection: directly add 10ul of the corresponding probe system concentrate to the two products obtained in the previous step to form 60 microliters of the 3WJ probe system containing:

10×Buffer 6 microliters

100×BSA 0.6 μl

10 micromolar probe A or A' 1.2 μl

10 micromolar probe B or B' 1.2 μl

ddH ₂ O 51 µl

The 3WJ conditions are: incubate at 37°C for 20 minutes, stop the reaction at 95°C for 5 minutes to detect fluorescence;

(4) Judgment of results: If there is fluorescence, there is a corresponding exon mutation.

A kit for detecting epidermal growth factor receptor exon 19, 21 mutations, at least including the following reagents:

(1) 2mM Mg ²⁺ , 200nM dNtP, 300nM upstream primers, 30nM downstream primers, Taq enzyme, and the corresponding 3WJ probes required to form the above asymmetric PCR system for amplifying EGFR19 exons E746~A750del 1×BSA, restriction endonuclease, probe A and probe B required by the system;

(2) 2mM Mg ²⁺ , 200nM dNtP, 300nM upstream primer, 30nM downstream primer, Taq enzyme required for the above-mentioned asymmetric PCR system for amplifying exon 21 L858R, and the corresponding 3WJ probe system Required 1X BSA, restriction enzymes, probe A' and probe B'.

The present invention has positive and beneficial effects:

Compared with the traditional gene mutation detection method, the method of the present invention has the advantages of faster, simpler, specific, sensitive and stable.

The method of the present invention organically integrates asymmetric PCR with nucleic acid constant temperature amplification and fluorescence detection technology: on the one hand, asymmetric PCR amplification technology and special primers are used to obtain a large amount of single-stranded DNA, which makes up for the 3WJ (constant temperature enzyme-cut nucleic acid detection technology) technology) inefficient linear amplification; on the other hand, the specificity and simplicity of the 3WJ technology is applied, so that the reaction temperature can be controlled at about 30 ^o C, without worrying about the reduction of enzyme activity, and no need for detection during the detection process Expensive and sophisticated temperature control equipment, special probe design makes it possible to distinguish as long as a simple fluorescence detection instrument.

The design features of the technical solution of the present invention make the technology not limited to one kind of restriction endonuclease, and there are more than one hundred kinds of restriction endonucleases on the market so far.

The technical solution of the present invention has two important innovations, (1) the nucleic acid hybridization detection area and the signal generation area are separated; (2) nucleic acid samples of various types and sources can be detected.

Description of drawings

Figure 1 is a configuration diagram of asymmetric PCR primers and probes for amplifying EGFR19 exon E746-A750del mutation.

Fig. 2 is a configuration diagram of asymmetric PCR primers and probes for amplifying the L858R mutation of exon EGFR21.

Fig. 3 is a graph showing sensitivity and specificity of the joint probe (JP) for detecting EGFR mutations. In the figure, A and B are the spectra for detecting EGFR L858R; C and D are the spectra for detecting EGFR 19DEL; A and C are the graphs of fluorescence intensity in the dynamic detection JP reaction system; B and D are the mini enzyme label when the reaction reaches the plateau A graph of the fluorescence intensity detected by the instrument.

Fig. 4 is a map of amplification and detection of EGFR mutation by APCR-JP method. In the figure, A and B are the electrophoresis graphs of APCR primers corresponding to L858R and 19DEL respectively, and the primer concentrations of lanes 1 to 8 are 1:1, 5:1, 10:1, 20:1, 50:1, 100:1, 200:1, 400:1, and 9 channels are PCR negative controls; it can be seen from the figure that when the primer concentrations are 5:1, 10:1, 20:1 and 50:1, it is easy to detect to ssDNA; C and D are fluorescence detection intensity maps, when the primer concentration is 10:1, the fluorescence intensity is the highest, L858R (C), 19DEL (D).

Fig. 5 is a comparison chart of detecting the content of L858R cells by the direct sequencing method (A) and the APCR-JP system of the present invention (B). It can be seen from the figure that the APCR-JP system can detect 5% of the mutant cells, while the direct sequencing method can only detect more than 20% of the mutant cells.

Detailed ways

The present invention is further described below in conjunction with specific examples.

Example 1 Extraction of Clinical Sample DNA

In this example, DNA is extracted from paraffin-embedded tissue sections of the lungs of patients with non-small cell lung cancer and quantified as a template for PCR detection. The paraffin-embedded tissue extraction kit from Takara Company was used, and the specific details are as follows:

(1) Extract sample DNA

① After cutting the paraffin-embedded tissue into 5 μm (4-10 μm) thick slices, use sterilized forceps to place 1-3 slices into a 1.5 ml Microtube equipped with TaKaRa DEXPAT®Easy, and the embedding tissue needs to be at least 6mm×6mm size. To prevent DNA contamination, sterilize the microtome with detergent and H ₂ O ₂ , then scrub with alcohol, do the same with the cutter and tweezers, and then irradiate with UV for more than 10 minutes.

②Cover the Microtube and heat at 100°C for 10 minutes. After heating for about 5 minutes, gently invert the Microtube 2 to 3 times and mix well.

Use Heat Blocks are very convenient.

The 1.5 ml Microtube is very hot, be careful not to burn yourself.

③ Immediately after heating, centrifuge at 17,000 rpm and 4°C for 10 minutes.

④ Immediately transfer the centrifuged Microtube to ice for 5 minutes.

⑤Use a pipette to absorb the water layer to avoid absorbing the paraffin film; when absorbing the water layer, you can see the jelly on the adsorption resin, and absorb the water layer on the jelly.

The aspirated aqueous layer can be used directly as a template for PCR reactions.

Use TaKaRa When common PCR enzymes such as Ex Taq® are used, the amount of DNA solution added is 1/10 of the total volume of the PCR reaction (5 μl is generally added to a 50 μl PCR reaction system).

When using MightyAmp® DNA Polymerase, which is highly resistant to blocking agents, for PCR reactions, the amount of DNA added is 4/10 of the total volume of the PCR reaction (20μl added to the 50μl PCR reaction system) can also obtain good amplification.

(2) Quantification: Quantify the extracted DNA with an ultraviolet spectrophotometer, and record the DNA concentration as 500ng/ul.

Example 2 Asymmetric PCR method to amplify clinical sample DNA

The DNA samples extracted in Example 1 were amplified using the primers and probes provided by the present invention. The asymmetric PCR primers and probes for amplifying EGFR19 exon E746～A750del mutations are shown in Figure 1 (see SEQ ID NO:1～4); wherein the asymmetric PCR for amplifying EGFR21 exon L858R mutations Primers and probes are shown in Figure 2 (see SEQ ID NO: 5-8).

The corresponding upstream primers and downstream primers were used to amplify EGFR19 exon E746～A750del and exon 21 L858R respectively, and the asymmetric PCR amplification system was composed as follows:

5X buffer 10 microliters

MgCl ₂ (25mM) 2.5μl

dNTP (10mM) 1 microliter

DNA template 2 microliters

Upstream primer (10 μM) 3 microliters

Downstream primer (10 μM) 0.2 μl

Taq DNA Polymerase (500 units) 0.3 µl

ddH ₂ O 31 µl

Total 50 microliters

The upstream and downstream primers are prepared in different proportions, respectively: 1:1, 5:1, 10:1, 20:1, 50:1, 100:1, 200:1 and 400:1, negative control group without DNA template.

See Table 1 for PCR amplification conditions:

Table 1 Asymmetric PCR amplification conditions

3WJ probe system: the above product is directly added to 10ul probe system concentrate to form a 60ul 3WJ probe system containing:

Buffer (10X) 6 microliters

BSA(100X) 0.6 μl

Probe A (10 μM) 1.2 μl

Probe B (10 μM) 1.2 μl

ddH ₂ O 51 µl

Total 60 microliters

The 3WJ conditions are: incubate at 37°C for 20 minutes, and stop the reaction at 95°C for 5 minutes.

The corresponding detection primers and probes were added to each detection system. The amplification primers and corresponding probe combinations are shown in Table 2. Fluorescence imaging plate reader (Gemini EM, USA) and mini single-channel fluorometer (OP- 162, Opulen Technology; China) to detect fluorescence.

In order to evaluate the specificity of the combined probe method, the corresponding artificial mutation template (MT) and wild-type mutation template (WT) shown in Table 2 were added to the above reaction system, that is, the method described in this example was used to The detection results are shown in Figure 3. This method can specifically detect the artificial mutant template, and the fluorescence intensity of the wild-type template is similar to that of the control group (A and C); the detection sensitivity, using the ten-step method to dilute the mutant template, JP The method can find the mutation template at 20nM level; A and B, detect EGFR L858R; C and D, detect EGFR 19DEL. A and C, the dynamic detection of the fluorescence intensity in the JP reaction system; B and D, the fluorescence intensity detected by the mini microplate reader when the reaction reaches the plateau.

Table 2 The combination of primers and probes used to detect 19 DEL and 21 L 858R mutations

At the same time, 12% polyacrylamide and SYBR Gold staining and gel imaging system were used to analyze the above PCR products, and the results are shown in Figure 4 A and B; the APCR-JP method amplifies and detects EGFR mutations, L858R(A) and 19DEL (B) APCR primers (as shown in Table 2); primer concentration (lanes 1 to 8) 1:1, 5:1, 10:1, 20:1, 50:1, 100:1, 200:1, 400:1, lane 9 is PCR negative control; when the primer concentration is 5:1, 10:1, 20:1 and 50:1, it is easy to detect ssDNA; when the concentration is 10 :1, the fluorescence intensity is the highest, L858R (C), 19DEL (D).

Example 3 Verification of the method of the present invention by direct sequencing

EGFR sequencing

(1) Primers (use the respective sequencing primers for 19DEL and L858R shown in Table 2)

(2) PCR system

Genomic DNA (>50ng/ul): 3ul

Forward primer (10uM) 3ul

Reverse primer (10uM) 3ul

dNTP mix (10uM) 1ul

5× buffer 10ul

Mg2 ⁺ (25mM) 5ul

Gota Taq (5U/ul) 0.3ul

ddH ₂ O 24.7ul

(3) PCR conditions - Cold-PCR, as shown in Table 3:

Table 3 PCR conditions

(4) PCR product purification

Axygen PCR Product Purification Kit.

(5) PCR sequencing reaction 10ul system:

Forward primer (3.2uM) 1 ul

PCR purification product: 3ul

Bigdye3.1: 1ul

5X buffer: 1.5ul

_ddH2O : 3.5ul

(6) Sequencing reaction conditions (see Table 4):

Table 4 Sequencing reaction conditions

(7) Sequencing: refer to the ABi3730XL sequencing process

(8) Results: As shown in Figure 5, the direct sequencing method (A) and the APCR-JP system (B) detected the content of L858R cells. The APCR-JP system can detect 5% of the mutant cells, while the direct sequencing method can only detect more than 20% of the mutant cells.

Example 4 Detection Kit for Epidermal Growth Factor Receptor Exon 19, 21 Mutations

According to the technical principles and specific technical solutions described in the content of the present invention, the following reagents can be collected to make commercialized detection kits for mutations in exons 19 and 21 of the epidermal growth factor receptor:

(1) 2mM Mg ²⁺ , 200nM dNtP, 300nM upstream primers, 30nM downstream primers, Taq enzyme, and the corresponding 3WJ probes required to form the aforementioned asymmetric PCR system for amplifying EGFR19 exons E746～A750del 1×BSA, restriction endonuclease, probe A and probe B required by the system;

(2) 2mM Mg ²⁺ , 200nM dNtP, 300nM upstream primer, 30nM downstream primer, Taq enzyme, and the corresponding 3WJ probe system required to form the aforementioned asymmetric PCR system for amplifying exon 21 L858R Required 1×BSA, restriction endonuclease, probe A' and probe B';

(3) Other available conventional biochemical reagents required in the detection process.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

SEQUENCE LISTING

<110> Chen Xiaoqi

<120> Primer probe system, method and kit for detecting epidermal growth factor receptor exon 19 and 21 mutations

<130> /

<160> 16

<170> PatentIn version 3.2

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<211> 22

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<213> Upstream Primer A

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atcccagaag gtgagaaagt ta twenty two

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<213> downstream primer B

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catcgaggat ttccttgttg 20

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<213> Probe A

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<213> Probe B

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<213> Upstream primer A'

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tgaaaacacc gcagcatgtc a twenty one

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<213> downstream primer B'

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<213> Probe A'

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<213> Probe B'

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agtaacggaa aaccgttact gstacatatt gatagcgacg 40

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<213> 19 DEL wild-type template

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ttcccgtcgc tatcaaggaa ttaagagaag caacatctcc gaaagc 46

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<213> 19 DEL mutant template

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<213> L 858R wild-type template

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Claims (2)

1. one kind is detected the primer probe system of ErbB1 9,21 exons mutations, comprise the asymmetric PCR primer that following amplification EGFR 19 exon E746～A750del sudden change and EGFR 21 exon L858R suddenly change, and the 3WJ probe matching with its primer:

。

2. detect a test kit for ErbB1 9,21 exons mutations, at least comprise following reagent:

(1) form the needed 2mM Mg of asymmetric PCR system of following amplification EGFR19 exon E746～A750del ²⁺, 200nM dNtP, 300nM upstream primer, 30nM downstream primer, Taq enzyme, and form the needed 1 × BSA of its corresponding 3WJ probe system, restriction restriction endonuclease, probe A and probe B:

5 × buffer, 10 microlitres

The MgCl that 25 millis rub ₂2.5 microlitre

DNTP 1 microlitre that 10 millis rub

The 10 micro-A of upstream primer as claimed in claim 13 microlitres that rub

The 10 micro-B of downstream primer as claimed in claim 1 0.2 microlitres that rub

Taq DNA Polymerase 0.3 microlitre of 500 units

DdH ₂o 33 microlitres

Amount to 50 microlitres

(2) form the needed 2mM Mg of asymmetric PCR system of following amplification 21 exon L858R ²⁺, 200nM dNtP, 300nM upstream primer, 30nM downstream primer, Taq enzyme, and form the needed 1 × BSA of its corresponding 3WJ probe system, restriction restriction endonuclease, probe A ＇ and probe B ＇:

5 × buffer, 10 microlitres

The MgCl that 25 millis rub ₂2.5 microlitre

DNTP 1 microlitre that 10 millis rub

10 micro-rub the A of upstream primer as claimed in claim 1 ＇ 3 microlitres

The 10 micro-B of downstream primer as claimed in claim 1 ＇ 0.2 microlitres that rub

Taq DNA Polymerase 0.3 microlitre of 500 units

DdH ₂o 33 microlitres

Amount to 50 microlitres;

In 60 microlitres, described 3WJ probe system contains:

10 × Buffer, 6 microlitres

100 × BSA, 0.6 microlitre

10 micro-probe A rubbing or A ＇ 1.2 microlitres

10 micro-probe B rubbing or B ＇ 1.2 microlitres

DdH ₂o 51 microlitres.