阅读说明：本技术 Snp位点集及胚胎核酸样本进行亲缘鉴定的方法和用途 (Method for carrying out genetic identification on SNP (Single nucleotide polymorphism) site set and embryonic nucleic acid sample and application ) 是由 李生斌 常辽 罗莉 李波 刘文嘉 于 2018-08-21 设计创作，主要内容包括：本发明涉及基因测序领域,具体涉及SNP位点集及胚胎核酸样本进行亲缘鉴定的方法和用途。所述SNP位点集包括SNP001～SNP200中的至少之一。本发明还提供了捕获探针、捕获芯片、引物组,以及基于胚胎核酸样本进行亲缘鉴定的方法和装置。本发明通过对胚胎进行亲权鉴定,可以减少病人移植错误带来的损害,而且具有高度准确性。(The invention relates to the field of gene sequencing, in particular to a method for carrying out genetic identification on an SNP locus set and an embryo nucleic acid sample and application thereof. The SNP site set includes at least one of SNP001-SNP 200. The invention also provides a capture probe, a capture chip, a primer group, and a method and a device for genetic identification based on the embryo nucleic acid sample. The invention can reduce the damage caused by the transplantation error of the patient by carrying out the paternity test on the embryo and has high accuracy.)

1. A set of SNP sites comprising at least one of the following SNP sites:

optionally, the set of SNP sites comprises any 50 SNP sites in table 1;

optionally, the set of SNP sites comprises any 80 SNP sites in table 1;

optionally, the set of SNP sites comprises any 100 SNP sites in table 1;

optionally, the set of SNP sites comprises any 120 SNP sites in table 1;

optionally, the set of SNP sites comprises any 150 SNP sites in table 1;

optionally, the set of SNP sites comprises any 180 SNP sites in table 1;

optionally, the set of SNP sites comprises all of the SNP sites in table 1.

2. A capture probe that specifically recognizes a predetermined nucleic acid sequence comprising at least one of the set of SNP sites of claim 1;

optionally, the capture probe is a single stranded oligonucleotide.

3. A capture chip carrying a plurality of capture probes according to claim 2, wherein each SNP site of the set of SNP sites of claim 1 has at least one corresponding capture probe.

4. A kit comprising at least one selected from the group consisting of:

the capture probe of claim 2, or

The capture chip of claim 3.

5. A method for genetic identification based on an embryonic nucleic acid sample, comprising:

(1) determining an embryo SNP typing result of the embryo and a parent SNP typing result to be detected of the parent to be detected aiming at the SNP locus set according to claim 1 based on an embryo nucleic acid sample and a parent nucleic acid sample to be detected;

(2) determining the genetic relationship between the embryo and the parent to be tested based on the embryo SNP typing result and the parent to be tested SNP typing result;

optionally, the embryo is from no more than 14 days after the formation of an in vitro fertilized egg;

optionally, the embryo is from no more than 7 days after the formation of an in vitro fertilized egg;

optionally, the embryo is from 3-6 days after the in vitro fertilized egg is formed;

optionally, the embryonic nucleic acid sample is from embryo culture fluid or ectoblast trophoblast single cells;

optionally, the nucleic acid sample is a whole genome;

optionally, the fetal nucleic acid sample is pre-amplified whole genome by at least one of:

multiple annealing circular amplification, multiple displacement amplification, degenerate oligonucleotide primer PCR.

6. The method of claim 5, wherein step (1) further comprises:

(1-1) constructing a sequencing library based on the embryonic nucleic acid sample and the parent nucleic acid sample to be detected;

(1-2) sequencing the sequencing library to obtain a sequencing result; and

(1-3) determining an embryo SNP typing result of the embryo and a parent SNP typing result to be tested of the parent to be tested based on the sequencing result;

optionally, step (1-1) further comprises:

fragmenting the whole genome sequence of the embryonic nucleic acid sample;

capturing the fragmentation treatment product using the capture probe of claim 2, or the capture chip of claim 3;

constructing a sequencing library by using the captured product as an insert;

optionally, the sequencing utilizes a sequencing platform of at least one of: hiseq4000, BGISEQ500 and Ion Proton.

7. The method of, wherein step (1-2) further comprises:

sequencing the sequencing library to obtain sequencing reads;

aligning the sequencing reads onto a reference genome, determining the location of the sequencing reads on the reference genome and the genotype of the SNP site;

and selecting SNP sites with the sequencing depth of more than 50, judging the SNP sites with the base occupying site total depth ratio of less than 0.1 or more than 0.9 as homozygous sites, and judging the SNP sites with the base occupying site total depth ratio of more than 0.3 and less than 0.7 as heterozygotes.

8. An apparatus for genetic identification based on an embryonic nucleic acid sample, comprising:

an SNP typing system, which is based on an embryo nucleic acid sample and a parent nucleic acid sample to be detected, aiming at the SNP locus set of claim 1, determining the embryo SNP typing result of the embryo and the parent SNP typing result to be detected of the parent to be detected;

the SNP typing comparison system is connected with the SNP typing system and is used for determining the genetic relationship between the embryo and the parent to be detected based on the embryo SNP typing result and the SNP typing result of the parent to be detected.

9. The device of claim 8, wherein the embryo is from no more than 14 days after the formation of an in vitro fertilized egg;

optionally, the embryo is from no more than 7 days after the formation of an in vitro fertilized egg;

optionally, the embryo is from 3-6 days after the in vitro fertilized egg is formed.

Optionally, the embryonic nucleic acid sample is from embryo culture fluid or ectoblast trophoblast single cells;

optionally, the nucleic acid sample is a whole genome;

optionally, the fetal nucleic acid sample is pre-amplified whole genome by at least one of:

multiple annealing circular amplification, multiple displacement amplification, degenerate oligonucleotide primer PCR.

10. The apparatus of claim 8 or 9, wherein the SNP typing system further comprises:

a library construction unit, wherein the library construction unit constructs a sequencing library based on the embryo nucleic acid sample and the parent nucleic acid sample to be detected;

the sequencing unit is connected with the library construction unit and is used for sequencing the sequencing library so as to obtain a sequencing result; and

the SNP typing determining unit is connected with the sequencing unit and determines the embryo SNP typing result of the embryo and the SNP typing result of the parent to be detected based on the sequencing result;

optionally, the sequencing utilizes a sequencing platform of at least one of: hiseq4000, BGISEQ500 and Ion Proton.

Technical Field

The invention relates to the field of gene sequencing, in particular to a method for carrying out genetic identification on an SNP (single nucleotide polymorphism) site set and an embryo nucleic acid sample and application thereof.

Background

More than 4000 million patients with infertility In China, and the IVF-ET (In vitro fertilization-Embryotransfer) technology can effectively solve the problem. The nationally approved domestic assisted reproductive organs exceed 400, and 10 ten thousand test-tube infants are estimated to be born in the year. But the IVF-ET is the characteristic of in vitro fertilization, so that illegal transactions such as buying and selling sperms, ova or embryos by illegal organizations or individuals and illegal surrogate pregnancy problems appear in the field. In addition, in the normal assisted reproductive organs, there are some reports of embryo implantation errors due to, for example, mishandling.

Further improvements are needed in how genetic identification can be performed for embryos.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, an object of the present invention is to provide a method and use for genetic identification of a set of SNP sites and an embryonic nucleic acid sample.

The inventors of the present invention found through research that: before the embryo is implanted into a human body, the paternity test is carried out on the embryo, on one hand, the paternity test on the embryo can be realized as early as possible, and on the other hand, the pain caused by error transplantation can be reduced. If the technology provided by the invention is applied to the test tube infant medical procedure, the paternity test before embryo implantation is added and a relevant report is issued, the market can be effectively standardized and the transplantation error can be avoided, and the technology has important significance for both patients and medical institutions.

To this end, according to a first aspect of the present invention, the present invention provides a set of SNP sites comprising: at least one of SNP001-SNP 200. Preferably at least 50 SNP sites of SNP001-SNP200 are included. As used herein, the term "set of SNP sites" refers to a set of sets comprising different SNP sites. According to the present example, the SNP site set is a set of SNP sites including any 50 or more SNP sites of 200 SNP sites, i.e., SNP001 to SNP200, as shown in Table 1. The "SNP site set" provided by the present invention may also be expressed as "a set of SNP sites", as required.

TABLE 1SNP sites

These SNP sites shown in Table 1 cover the entire genome, and as many accurate typing sites as possible can be obtained by sequencing these SNP sites. By using these SNP sites, accurate typing results can be obtained by performing SNP typing on nucleic acid samples of embryos at embryonic stages. According to the rs number corresponding to each SNP locus, the detailed information of the corresponding SNP locus can be obtained by utilizing the hg of the human reference genome.

According to the embodiment of the present invention, the SNP site set provided by the present invention may include any 50 SNP sites in table 1; or may include any of the 80 SNP sites in table 1; or may include any 100 of the SNP sites in table 1; or may include any of the 120 SNP sites in table 1; or may include any of the 150 SNP sites in table 1; or may comprise any of the 180 SNP sites in table 1; preferably, all SNP sites in Table 1 may be included. When the number of SNP sites included in the provided SNP site set is larger, the more accurate the result of SNP typing can be obtained. According to embodiments of the present invention, when the provided SNP site set includes at least 50 SNP sites in table 1, accurate SNP typing results can be obtained for paternity testing before embryo implantation.

According to a second aspect of the invention, there is provided a capture probe which specifically recognizes a predetermined nucleic acid sequence comprising at least one of the set of SNP sites according to the first aspect of the invention.

According to an embodiment of the invention, the capture probe is a single-stranded oligonucleotide. By preparing the gene chip containing the site, synthesizing a single-stranded oligonucleotide probe through reverse transcription, establishing a library and sequencing through a liquid phase hybridization capture system, any SNP site in SNP001-SNP200 can be conveniently and quickly captured by using the chip.

According to a third aspect of the present invention, there is provided a capture chip carrying a plurality of capture probes, the capture probes being according to the second aspect of the present invention, and at least one corresponding capture probe for each SNP site of the set of SNP sites according to the first aspect of the present invention.

According to a fourth aspect of the invention, there is provided a kit comprising at least one selected from the group consisting of: a capture probe according to the second aspect of the invention; the capture chip according to the third aspect of the invention.

According to a fifth aspect of the present invention, there is provided a method for genetic identification based on an embryonic nucleic acid sample, comprising: (1) based on an embryo nucleic acid sample and a parent nucleic acid sample to be detected, aiming at the SNP locus set of the first aspect of the invention, determining an embryo SNP typing result of the embryo and a parent SNP typing result to be detected of the parent to be detected; (2) and determining the genetic relationship between the embryo and the parent to be tested based on the embryo SNP typing result and the parent to be tested SNP typing result. The invention determines the genetic relationship between the embryo and the parent to be detected by carrying out SNP typing on the embryo nucleic acid sample, carrying out SNP typing on the parent nucleic acid sample to be detected and comparing the embryo SNP typing result with the SNP typing result of the parent to be detected.

According to the embodiment of the present invention, the above method for performing genetic identification based on an embryonic nucleic acid sample may further have the following technical features:

according to an embodiment of the invention, the embryo is from no more than 14 days after the formation of the in vitro fertilized egg. According to an embodiment of the invention, the embryo is from no more than 7 days after the formation of the in vitro fertilized egg. The trophoblast cells of the blastocyst can be obtained within 7 days, or the SNP typing of the embryo can be realized by utilizing the free circulating DNA in the embryo culture solution.

According to an embodiment of the invention, the embryo is from 3-6 days after the in vitro fertilized egg is formed.

According to embodiments of the invention, the embryonic nucleic acid sample is derived from embryonic culture fluid or blastocyst stage trophectoderm single cells. Paternity test is carried out before embryo implantation, 1 cell of blastocyst trophectoderm is taken through a slight cutting technology, or free circulating DNA (cfDNA) in a culture solution from fertilized eggs to the third day of cleavage stage or the fifth day of blastocyst stage is utilized, after whole genome amplification, gene chips formed by gathering SNP sites for individual identification are utilized to carry out capture, library construction and sequencing, and genetic information of parents and embryos is obtained through biological information analysis so as to confirm paternity.

According to an embodiment of the invention, the nucleic acid sample is a whole genome. By sequencing the whole genome of a nucleic acid sample, the problem that a trace sample does not meet the initial amount of library construction can be solved, and the pg-grade DNA can be amplified to the mu g grade.

According to an embodiment of the invention, the fetal nucleic acid sample is pre-amplified whole genome by at least one of: multiple annealing circular amplification, multiple displacement amplification, degenerate oligonucleotide primer PCR. The whole genome is amplified, so that the amplification of a single cell genome (5-7pg DNA) and even a single chromosome can be realized, wherein the whole genome amplification of trace DNA can be conveniently and quickly realized by applying technologies such as multiple annealing and cloning-based amplification cycles (MALBAC), Multiple Displacement Amplification (MDA) and degenerate oligonucleotide primer PCR (DOP-PCR). In the MALBAC technology, random primers are hybridized with template DNA, and the template is amplified by using strand displacement polymerase at about 65 ℃ to generate a 'semi-amplicon'. Subsequent amplification cycles produce a complete amplicon that forms a hairpin structure, preventing itself from becoming a template. In this way, it is avoided that the amplicon becomes the template and only the original template is used, thereby reducing the amplification error rate. The MDA utilizes a random six-base primer to anneal with template DNA at a plurality of sites, and then Phi29DNA polymerase with high amplification efficiency and fidelity simultaneously initiates replication at a plurality of sites of the DNA, and the Phi29DNA polymerase has strong template binding capacity for the template and can continuously amplify a 100kb DNA template without dissociating from the template. Meanwhile, the enzyme has 3 '-5' exonuclease activity, so that high fidelity of amplification is ensured. The DOP-PCR primer is designed to have a 3 'end as a binding site with high frequency in a genome, a continuous degenerate base in the middle and a 5' end as an about 10-base fixed sequence, the genome is randomly broken to prepare an OminiPLex library, a series of short and overlapped templates are formed, and the product is about 400bp and has the quality of mu g grade.

According to an embodiment of the present invention, the step (1) further comprises: (1-1) constructing a sequencing library based on the embryonic nucleic acid sample and the parent nucleic acid sample to be detected; (1-2) sequencing the sequencing library to obtain a sequencing result; and (1-3) determining the embryo SNP typing result of the embryo and the parent SNP typing result to be detected of the parent to be detected based on the sequencing result.

According to an embodiment of the present invention, the step (1-1) further includes: fragmenting the whole genome sequence of the embryonic nucleic acid sample; capturing the fragmentation treatment product, wherein the capture is performed by using a capture probe according to the second aspect of the invention or a capture chip according to the third aspect of the invention; using the captured product as an insert to construct a sequencing library.

According to an embodiment of the invention, the sequencing utilizes a sequencing platform of at least one of: hiseq4000, BGISEQ500 and Ion Proton. The preparation method of the sequencing library is carried out according to the requirements of the selected sequencing method, the sequencing method can select but not limited to Hisq2000/2500 sequencing platform of Illumina, Ion Torrent platform of Life Technologies, BGISEQ platform of BGI and single molecule sequencing platform according to the difference of the selected sequencing platform, the sequencing mode can select single-ended sequencing or double-ended sequencing, and the obtained off-line data is a sequencing and reading fragment called reads (reads).

According to an embodiment of the present invention, the step (1-2) further includes: sequencing the sequencing library to obtain sequencing reads; aligning the sequencing reads onto a reference genome, determining the location of the sequencing reads on the reference genome and the genotype of the SNP site; and selecting SNP sites with the sequencing depth of more than 50, judging the SNP sites with the base occupying site total depth ratio of less than 0.1 or more than 0.9 as homozygous sites, and judging the SNP sites with the base occupying site total depth ratio of more than 0.3 and less than 0.7 as heterozygotes. For the single cell separated from the embryo or the situation that the cfDNA in the culture solution has the condition of allele tripping and inaccurate amplification preference amplification and other types after the whole genome amplification, when the SNP is typed, the typing standard of homozygous and heterozygous is modified when the SNP locus is judged to be homozygous or heterozygous, and the expression is as follows: for SNP sites with a sequencing depth of more than 50, sites with a base ratio of less than 0.1 or more than 0.9 are considered as homozygous sites, and sites with a base ratio of more than 0.3 and less than 0.7 are considered as heterozygous sites. For example, if the total depth of sequencing is 1000 x (i.e., the number of reads comprising the site is 1000) using SNP0001, i.e., the rs1005533 site, wherein the ratio of the G bases to the total depth of sequencing is 0.6 if the measured G bases is 600 x and the a bases is 400 x, the SNP site is determined to be a heterozygote if the ratio of the obtained bases is greater than 0.3 and less than 0.7. Thus, on the one hand, SNP typing results can be obtained by measuring a plurality of SNP sites, and on the other hand, when SNP sites are judged to be homozygous or heterozygous, precise SNP typing results can be obtained strictly according to the above criteria.

According to a sixth aspect of the present invention, there is provided an apparatus for genetic identification based on an embryonic nucleic acid sample, comprising: the SNP typing system is based on an embryo nucleic acid sample and a parent nucleic acid sample to be detected, and determines an embryo SNP typing result of the embryo and a parent SNP typing result to be detected of the parent to be detected aiming at the SNP locus set according to the first aspect of the invention; the SNP typing comparison system is connected with the SNP typing system and is used for determining the genetic relationship between the embryo and the parent to be detected based on the embryo SNP typing result and the SNP typing result of the parent to be detected.

According to the embodiment of the invention, the above apparatus for genetic identification based on fetal nucleic acid samples may have further additional technical features:

according to an embodiment of the invention, the embryo is from no more than 14 days after the formation of the in vitro fertilized egg.

According to an embodiment of the invention, the embryo is from no more than 7 days after the formation of the in vitro fertilized egg.

According to an embodiment of the invention, the embryo is from 3-6 days after the in vitro fertilized egg is formed.

According to embodiments of the invention, the embryonic nucleic acid sample is derived from embryonic culture fluid or blastocyst stage trophectoderm single cells.

According to an embodiment of the invention, the nucleic acid sample is a whole genome.

According to an embodiment of the invention, the fetal nucleic acid sample is pre-amplified whole genome by at least one of: multiple annealing circular amplification, multiple displacement amplification, degenerate oligonucleotide primer PCR.

According to an embodiment of the present invention, the SNP typing system further includes: a library construction unit, wherein the library construction unit constructs a sequencing library based on the embryo nucleic acid sample and the parent nucleic acid sample to be detected; the sequencing unit is connected with the library construction unit and is used for sequencing the sequencing library so as to obtain a sequencing result; and the SNP typing determining unit is connected with the sequencing unit and determines the embryo SNP typing result of the embryo and the SNP typing result of the parent to be detected based on the sequencing result.

According to an embodiment of the invention, the sequencing utilizes a sequencing platform of at least one of: hiseq4000, BGISEQ500 and Ion Proton.

The beneficial effects obtained by the invention are as follows: (1) the method provides a detection scheme for paternity test of embryos for the first time, solves the huge damage of spirit and economy caused by transplantation errors of patients, and has great significance for standardizing test-tube infant procedures by giving paternity test reports of corresponding parents before embryo implantation.

(2) High accuracy. The invention selects a large number of SNP sites based on a Chinese polymorphic site database in a thousand-person genome plan, the number of SNP sites is large, the whole genome is uniformly covered, the accuracy is high, and the non-paternal exclusion rate can reach 99.99999999%. In the early embryo in-vitro culture process, a trace amount of cfDNA is released into a culture medium due to apoptosis, and the content of the cfDNA in the culture solution in the blastocyst stage is higher than that in the cleavage stage and is about dozens of pg. High-depth sequencing of a large number of SNP sites can improve the number of polymorphism sites for accurate typing, thereby judging the paternity.

(3) The method reduces the 5% false positive of the typing result of the single cell sequencing data by the current SNP typing method of the high-throughput sequencing data to 1%.

Drawings

FIG. 1 is a schematic diagram of an apparatus for genetic identification based on an embryonic nucleic acid sample according to an embodiment of the present invention.

FIG. 2 is a schematic diagram of a SNP typing system provided according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

When the paternity test is carried out on the fetus, the paternity test of the fetus usually needs to be carried out within 6-8 weeks after the embryo is implanted into the mother, and how to carry out the test on the paternity test of the fetus can reduce the huge damage of embryo implantation errors to the heart of a person concerned at an earlier stage before the embryo is not implanted into the mother, such as the identification of a fertilized egg or the identification of the embryo which is still in the cleavage stage.

Moreover, as mentioned above, according to the ethical guidelines for the study of human embryonic stem cells, published by the department of science and technology and the department of health of the national institute of 2004, guidelines are given for the study of human embryonic stem cells, one of which is to comply with the "blastocyst obtained by in vitro fertilization, somatic cell nuclear transfer, a parthenocarpy technique or genetic modification, the in vitro culture period of which is not more than 14 days from the start of fertilization or nuclear transfer". When the invention is used for identifying the paternity of the embryo, the embryo in the cleavage stage or the blastocyst within 14 days can be cultured, and the culture solution or single cells are taken for SNP typing, so that the SNP typing result can be obtained and meets the requirements.

The inventor creatively thinks that the pain caused by error transplantation is reduced by using a single cell or carrying out genetic identification on a nucleic acid sample of an embryo in a culture solution. However, the traditional paternity test technology and noninvasive prenatal paternity test usually adopt blood spots, hairs, oral swabs and the like of individuals, the demand for test materials is higher than 100pg, and the cfDNA content in a single cell or embryo culture solution is usually only about 10 pg. How to obtain the nucleic acid information of the embryo through a small amount or trace amount of cfDNA and reduce the false positive rate caused by sequencing at the same time to obtain a typing result with high accuracy for paternity test is very important.

Therefore, the invention screens and obtains an SNP site set containing 200 SNP sites, including at least 50 sites in SNP001-SNP200, by using the statistical analysis of SNP data of Han nationality population, as shown in Table 1. These sites cover the entire genome and high depth sequencing through these SNP sites can yield as many accurate typing sites as possible. By using the SNP sites, the accuracy of amplification of cfDNA in embryo culture solution at different stages or single cells at the blastocyst stage is high.

In this context, the expression "embryo culture fluid" or "embryo culture medium" refers to: a substrate or fluid for culturing the embryo in vitro.

In this context, the expression "cfDNA" refers to free DNA released from an embryo into an embryo culture fluid or medium during the in vitro culture of the embryo.

According to the embodiment of the invention, fertilized eggs obtained by in vitro fertilization-embryo transfer (IVF-ET) or intracytoplasmic sperm injection (ICSI) method are cultured to the blastocyst stage, and single cells are obtained by a Preimplantation Genetic Screening (PGS) process, and are frozen at-20 ℃. For example, at 5-6 days after insemination, the blastocyst is sufficiently expanded, and the blastocyst is harvested at a site distant from the inner cell mass.

According to an embodiment of the invention, the embryo culture fluid is a drop of culture that cultures the embryo to the blastocyst stage or the cleavage stage. The key point is to collect the culture solution and completely remove the granular cells, so that no exogenous DNA interference exists in the culture solution.

According to another aspect of the present invention, the present invention provides an apparatus for genetic identification based on an embryonic nucleic acid sample, as shown in FIG. 1, comprising an SNP typing system and an SNP typing and comparing system, wherein the SNP typing and comparing system is connected with the SNP typing system; the SNP typing system is based on an embryo nucleic acid sample and a parent nucleic acid sample to be detected, and aiming at the SNP site set, the embryo SNP typing result of the embryo and the parent SNP typing result to be detected of the parent to be detected are determined; and the SNP typing comparison system is used for determining the genetic relationship between the embryo and the parent to be detected based on the embryo SNP typing result and the parent to be detected SNP typing result.

According to an embodiment of the present invention, the SNP typing system is shown in fig. 2, and further includes: the device comprises a library construction unit, a sequencing unit and an SNP typing determination unit; the sequencing unit is connected with the library construction unit, and the SNP typing determination unit is connected with the sequencing unit; the library construction unit constructs a sequencing library based on the embryo nucleic acid sample and the parent nucleic acid sample to be detected; the sequencing unit is used for sequencing the sequencing library so as to obtain a sequencing result; and the SNP typing determining unit determines the embryo SNP typing result of the embryo and the SNP typing result of the parent to be detected based on the sequencing result.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.