JPH04108385A - Production of equalized cdna library - Google Patents

Abstract

PURPOSE:To prepare an equalized cDNA library by separating cDNA reassociated into double strands and cDNA of intact single strands, synthesizing the single-stranded cDNA into double-stranded cDNA and respectively cloning the cDNA. CONSTITUTION:A double-stranded cDNA synthesized by using an mRNA as a template is denatured and further reassociated and the reassociated cDNA is separated from the cDNA of intact single strands. The resultant separated single-stranded cDNA is synthesized into a double-stranded cDNA and respectively cloned to prepare an equalized cDNA library. Furthermore, the single-stranded cDNA is preferably previously provided with an adhesive end at the 3'-terminal thereof and fragmented and the cDNA to be cloned or a fragment thereof is preferably amplified by a polymerase chain reaction(PCR) method. The single- stranded cDNA remaining after the reassociating reaction can be further amplified by the PCR method to readily afford the cDNA in an amount sufficient to ensure an excellent cloning efficiency.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) This invention is directed to the production of homogenized cDNA libraries (equal cDNA libraries).
ized cDNA library) and its production method. More specifically, this invention is applicable to a wide range of research fields such as medicine and biology, such as cloning of unknown genes, identification of transcribed regions on genome sequences or cosmid clones, and simultaneous identification of expression patterns of multiple gene groups. The present invention relates to a homogenized cDNA library useful for and a method for producing the same.

(Prior Art) In recent years, scientific elucidation of the tissues and mechanisms that make up living organisms and accompanying technological applications have progressed rapidly, and the foundations of genetic engineering are being established one after another.

Under these circumstances, genes from all living things, from prokaryotes to eukaryotes including humans, have been identified with structures or functions unique to species and individual tissues, and through analysis of their genetic information, A large amount of knowledge has been accumulated, and on the other hand, storing the individual genes obtained in this way so that they can be used at any time as needed is important for further information about that gene. This is an extremely important issue from the viewpoint of analysis and its medical, biological, and engineering applications.

Traditionally, when storing a specific gene, a method has been used in which the genetic DNA is partially restricted and digested, each fragment is cloned into a cloning vector, etc., and a so-called gene library is created from a collection of individual clones. It is being

One such gene library is a genomic library. This is the fragmentation and individual cloning of all the genes (genomic DNA) present in a particular organism.

It is estimated that less than 1% of a particular organism's genome is coated with proteins, that is, the portion that plays an important role in actually forming the organism. That is, most of the information in the library is often unnecessary. A cDNA library is created by cloning only the portion encoding this protein.

This process uses reverse transcriptase as a template to generate complementary DNA (complementary DNA) in both prokaryotes and eukaryotes, using mRNA present in cells as a template.
: cDNA) is synthesized and individually cloned to create a gene library.

This library can be used in a variety of ways that genomic libraries cannot, such as connecting it to an expression vector and expressing the protein in animal cells, and can be widely produced in many different types of cells from more species than before. It has been done.

(Problems to be Solved by the Invention) However, unlike genomic libraries, cDNA libraries can be of different types depending on which cells the material is taken from. This is because most multicellular organisms have specialized individual cells, or in other words, are composed of aggregates of various different types of cells. Moreover, each individual cell has a set of genes, or the group of genes to be expressed is determined depending on the type of cell, and only the expressed genes are transcribed into mRNA.

Furthermore, one cell has many types of expressed genes; for example, in the case of liver cells, it is estimated that the number of types of expressed genes reaches 20,000 to 30,000. Moreover, each of them does not exist in the same proportion; in the case of hepatocytes,
Approximately 16% of the amount of mRNA is one type of mRNA that coats a protein called albumin. Furthermore, when chicken oviduct cells are stimulated with nistrogien, 50% of the total amount of mRNA transcribed is one type of mRNA called ovalbumin. On the other hand, it is estimated that the gene with the lowest content is only 0.0005% or less of the total mRNA, and the difference is actually 10,000 times greater.

Therefore, in the conventional method, that is, when cDNA is synthesized using mRNA transcribed from an expressed gene as a template and a cDNA library is created by individually cloning them, the difference in the content of each mRNA species is This is directly reflected in the difference in the content rate of each type of clone making up the library. In other words, in the case of a hepatocyte cDNA library, approximately 16% of the total is cDNA of the same species.
clone, while the lesser one is only 0,00
This means that only 0.5% of them exist.

For this reason, a library that also includes cDNA species with a low content rate becomes large.

For example, in the liver, the total number of all types of mRNA is at most a small number, but conventional libraries do not contain all of the mRNA unless it is several dozen to several million in size.

When using a cDNA library, what is actually required is one cDNA for one type of gene, and cDNAs that are duplicated in the library are not only redundant but also difficult to obtain the desired c[)N. , it also hinders the search for A. Of course, even in conventional cDNA libraries containing many such extra cDNA clones,
When targeting a specific gene whose nucleotide sequence is known, it is relatively easy to obtain the target cDNA using known methods such as hybrid motion probing.
can be identified and isolated. Furthermore, even if the base sequence is unknown, screening methods have been established for cases where antibodies against the protein exist.

The problem is identifying and isolating cDNA encoding the protein using function as an indicator.

In this case, each cDNA must be inserted into an expression vector, the protein produced within the cell, and the effect of that protein on the cell must be investigated. Also,
There are many genes that cannot be identified or isolated unless a huge number of clones in a library are searched one by one.

In recent years, has the use of cDNA libraries to search for unknown genes become more important?
The presence of large numbers of duplicate and redundant cDN,A in conventional cDNA libraries is a major problem.

Also, for the same reason, although the types of genes that can be expressed are limited (it is said that there are about 100,000 types in humans), it is important to have one set of cDNA types. I haven't even been able to do it.

If it were possible to create such a library, there would no longer be a need for each individual researcher to create a cDNA library for each case.

This invention was made in view of the above circumstances, and it overcomes the problems of conventional cDNA libraries and provides a homogenized cDNA library that contains the molecular species of each mRNA, that is, the expressed gene, in a more equal proportion. The purpose of this invention is to provide a method for producing a DNA library and a homogenized cDNA library.

(Means for Solving the Problems) The present invention solves the above problems by using mRNA
The double-stranded cDNA synthesized using A as a template is denatured and further re-associated, the cDNA that has re-associated into double-strands and the cDNA that remains single-stranded are separated, and this single-stranded cDNA is converted into double-stranded cDNA. A method for producing a homogenized cDNA library, characterized in that it is synthesized into cDNA and cloned individually;
A homogenized cDNA library prepared by such a method is provided.

This invention also provides a preferred embodiment of fragmenting a single-stranded cDNA by providing a cohesive end in advance at its 3' end, and amplifying the cDNA to be cloned or a fragment thereof by PCR. There is.

The configuration of this Q invention will be explained in detail below.

First, in this invention, a cDNA library of the gene can be created for any type of cell of any species.

In principle, this also involves the creation of a cDNA library containing a complete set of genes that can be expressed for each species.

Furthermore, extraction and purification of mR'NA, which serves as a template for cDNA, can be carried out according to known conventional methods, for example, as follows.

That is, for example, since eukaryotic cytoplasmic mRNA is concentrated in polysomes, which are complexes with liposomes, the polysomes are first separated by differential centrifugation or centrifugation in a sucrose solution or its density gradient. Next, mRNA is extracted from the polysome using a mixture of phenol and chloroform, phenol saturated with a pH 9 buffer, hot 5DS-phenol, or the like.

The mRNA extracted in this way is further analyzed by size-specific mRNA using sucrose density gradient centrifugation, gel electrophoresis, etc.
Purify to NA.

Known methods can also be used to synthesize cDNA from the mRNA thus obtained. for example,
Most of the mRNA present in the cytoplasm of eukaryotic cells is
' Since the terminal has a polyA sequence consisting of ioo to 200 bases, for example, an oligo (dT) primer is annealed to this polyA, and this is used as a primer to generate a polynucleotide consisting of NA that is complementary to the RNA strand using reverse transcriptase. Synthesize nucleotides. Furthermore, this polynucleotide is treated with alkali to degrade the RNA strand and create a single-stranded cDNA.
However, a hairpin loop is formed at the 3' end of this single-stranded cDNA, and a complementary cDNA strand is synthesized using this as a primer to form a double-stranded cDNA.

When creating a regular cDNA library,
Both ends of this double-stranded cDNA are treated with, for example, S1 nuclease to add cohesive ends, and then cloned using an appropriate loning vector. In the present invention, all of the obtained cDNA is Rather than cloning them, they are selected and homogenized to have equal proportions of each cDNA species.

In order to achieve such homogenization, first, all of the obtained double-stranded cDNAs are denatured (denatured) by, for example, immersing them in an alkaline denaturing solution or by applying heat.
tur'ation) to dissociate into single strands, and then this single stranded cDNA is reacted in a reassociation buffer at an arbitrary temperature (for example, 65°C) to reassociate (reassociate) into double stranded cDNA. reassoc 1ation). At this time, each single-stranded cDNA naturally re-associates complementary cDNAs, or cDNAs present in large numbers in duplicate in the reaction solution.
DNA quickly re-associates, resulting in low cDNA content.
A needs time to complete the reconvening. Therefore, when the reassociation reaction is stopped at an appropriate time, the reaction mixture contains cDNA that has reassembled into double strands and cDNA that remains single stranded.
will exist. Among these, most of the double-stranded cDNA groups are cDNAs that originally existed in large numbers in duplicate.
The remaining single-stranded cDNA is occupied by the NA species.
In this group, there are single strands of cDNA species with a high content that have failed to reassociate within a certain period of time, and single strands of cDNA species with a low content that are originally difficult to reassociate. Therefore, the double-stranded cDNA and single-stranded cDNA present in the reaction solution
If A is separated using a known method, such as a hytroxylapatite column, and only the single-stranded cDNA is synthesized again into double-stranded cDNA in the same manner as described above, in this group of cDNAs, each cDNA species will be separated. The difference in the content of each type becomes significantly smaller.

Furthermore, by repeating such denaturation and reassociation processes multiple times, it is possible to finally obtain a collection of cDNAs containing equal proportions of each cDNA species, and these cDNAs are inserted and ligated into cloning vectors according to standard methods. By doing so, a homogenized cDNA library can be created.

The denaturation and reassociation process itself used to achieve such homogenization can be used, for example, when selecting mRNA that is specifically expressed in only one cell from two types of cell mRNA, or when using an arbitrary method. This method is widely used to select mRNA whose expression is specifically induced by such means (for example, by applying a specific hormone) from mRNA expressed uniquely to that cell. for example,
When creating a cDNA library containing only mRNA species that are specifically expressed in cell (A) using cell (A) and cell (B), generally the mRNA of cell (A) is converted into single-stranded cDNA. A procedure is being taken to clone the remaining single-stranded cDNA by combining it with the mRNA of the cell (B), but at this time, the cDNA of both cells (A) and (B) is In order to completely remove commonly expressed mRNA species, it is necessary to add a large excess of mRNA from cells (B) to cDNA from cells (A). However, it is essential that none of the complementary strands of the cDNAs are dropped and that they remain in a uniform ratio, so it is an absolute condition that the complementary strands of each cDNA subjected to the reassociation reaction must be in an exact one-to-one ratio. .

This also allows all mRNA species (c DNA species)
It is theoretically guaranteed that all of the following are included in the homogenized library.

However, when producing a homogenized cDNA library by the above method, there is a risk that the following two problems may occur depending on the type of target cells.

One is the shedding of necessary CDNA species during the denaturation and reassociation process for homogenization. Usually single-stranded cDN
When A is subjected to an association reaction, those whose sequences are complementary associate with each other to form double-stranded cDNA. However, in reality, even if single strands of cDNA are derived from different mRNAs, if they each have a homologous region of about 50 bp, there is a possibility that the single strands of cDNA may form a double strand. There is. For example, there are about 10 types of mRNA that encode the protein actin, and their protein-coding regions are highly homologous, so when the single-stranded cDNAs of each of these are combined, they code for actin in the same way. However, cDNAs of different types may form double strands. As described above, in the present invention, CDNAs that have formed double strands as a result of reassociation reaction within a certain period of time are excluded from the library. Therefore, when different types of single-stranded cDNAs re-associate as in the case of the actin gene described above, an incomplete library is created in which several types of necessary cDNAs are missing. In order to overcome such problems, in the present invention, full-length cDNA synthesized from mRNA is fragmented in advance into several hundred base pair units by mechanical shearing force, and the 3′
A method for producing a homogenized cDNA library by cloning only cDNA fragments in the terminal region is also provided.

In other words, in general, regions of high homology with other genes on a single mRNA are limited to only a few, such as the protein coding region, and conversely, the untranslated region at the 3' end has its own specificity. is known to be high.

Therefore, cDNAs in the 3' end region will not form double strands between different types of single-stranded cDNAs even through reassociation reactions, and there is no risk that necessary cDNA species will drop out of the library.

In this method, when selectively cloning cDNA fragments in the 3'-end region, for example, before fragmenting the full-length cDNA, the fragments can be cloned by attaching cohesive ends to the 3-end. It is only necessary to enable ligation to the cloning vector.

Next, another problem with the production method of this invention is that as a result of removing and homogenizing most of the redundant CDNA species that exist in large numbers, the cDNA that is finally used for cloning is
This means that because the amount of A decreases, the efficiency of cDNA integration into the cloning vector decreases. However, such problems arise when cloning target c
PCR method (Polymerase Chain)
This can be solved by amplifying the problem using a method such as Reaction). For example, if the single-stranded cDNA remaining after the reassociation reaction is amplified by PCR, a sufficient amount of cDNA can be obtained to ensure good or loning efficiency.
NA can be easily obtained.

Therefore, in the example shown below, an example will be shown in which a homogenized cDNA library was prepared using Ltk- cells, which are a mouse fibroblast cell line.

Example 1 (mRNA purification and cDNA synthesis) Ltk- cell mRNA was extracted and purified, and was synthesized into cDNA.

First, Ltk- cells were cultured in a medium containing 1O% fetal serum, and total RNA was isolated from the growing cells according to a conventional method. Next, from these RNAs, mR having a polyA sequence was purified using an mRNA purification kit (Pharmacia).
NA was extracted and purified.

Furthermore, mRNA of Ltk- cells prepared in this way
In A, mRNA of neo gene (1,3 kb) and tk gene (1,8 kb), which are foreign genes, were used as control markers at 10 w/w% and 0.0005 w, respectively.
/w% was added and used as a template for cDNA.

5 μg of the mRNA thus obtained was added according to a conventional method.
Oligo(dT)-Not I primer (5=-A
ATTCGCGGCCGCTTTTTTTTTTT
cDNA was synthesized using TT-3-Promega).

Next, the synthesized full-length double-stranded cDNA was
The fragments were fragmented into 200-400 bp fragments using 5onifier 250 (Branson), selected by agarose gel electrophoresis, and then treated with T4 DNA polymerase.

Example 2 (Amplification of CDNA fragment by PCR method) The double-stranded CDNA fragment obtained in Example 1 was amplified using PCR method.

Since both ends of each CDNA fragment obtained in Example 1 are plant ends, the following linker-primer (LL-
R1) was attached.

LL-RIA: 5-- pGAGATATTAGA
ATTCTACTC-3LL-RIB・3--T
ATAATCTTAAGATGAGp-5This LL-R
Since the protruding ends of CDNA 1 do not adhere to each other, a single molecule of the linker specifically binds to both ends of the CDNA fragment.

After attaching LL-RI to both ends of the cDNA in this way, the LL-RIA oligomers shown in FIG.
PCR was performed using this as a primer to amplify the CDNA fragment. That is, log cDNA was added to a 100 μm reaction solution, and a cycle of 2 minutes at 94° C., 2 minutes at 50° C., and 2 minutes at 72° C. was repeated 25 times to amplify the cDNA.

Next, the amplified cDNA was extracted with an equal volume of phenol-chloroform-isoamyl alcohol saturated with TE, and further extracted with Centricon-100 (Amicon).
Free primers that were not incorporated into the cDNA strand were removed using .

Example 3 (Uniformization of cDNA) The cDNA fragments amplified in Example 2 were subjected to denaturation and reassociation treatment three times to equalize the content of each cDNA species.

That is, in the first homogenization (EI) and the second homogenization (E II), 20 Mg of amplified cDNA was
．． 5 ynl IOμ in Eppendorf tube
Dissolve in 1 liter of distilled water and add an equal amount of 2x hybridi to this.
zation solution (0,24M Na H2P O4
[pH6,8], 1.64M NaCl, 2mMED
TA, 0.2% 5DS) was added. In addition, in the third homogenization (EI[), 100 μg was added to the same amount of solution as above.
cDNA was added.

In order to prevent evaporation of each sample solution, the surface thereof was covered with light oil and boiled for 5 minutes to denature the cDNA and make it a single strand. Next, EI and EI[ were subjected to a reassociation reaction at a temperature of 65° C. for 12 hours, and EI[[ was subjected to a reassociation reaction at the same temperature for 24 hours.

As an indicator of the degree of reassociation of complementary NA strands,
There is a Cot value, but Co when making El, Ell, and Em.
The t values (mol, 1itre=, sec) are 130, 130. It was 1100.

As a result of such denaturation and reassociation, cDNA that had reassociated into double strands and cDNA that remained single stranded were obtained, and these were incubated at a temperature of 60°C filled with hytroxylapatite according to a conventional method. After separating in a column and desalting single-stranded cDNA fragments using Centricon-100, these were amplified by the same PCR method as in Example 2 and synthesized into double-stranded fragments again.

Example 4 (Preparation of cDNA library and evaluation of its homogenization) CDNA subjected to homogenization treatment three times in Example 3
The fragment was cloned and c
A DNA library was created.

First, the double-stranded cDNA obtained in Example 3 was treated with restriction enzyme Eco.
Cut with RI and NotI, and convert this into the plasmid vector pBluescript SK (-) with EcoRI.
- Insert ligated into the Not I cleavage site.

Furthermore, this vector was introduced into host cells, and the host cells were transformed to form colonies.

Next, the homogeneity of the cDNA library thus obtained was evaluated by the following method. In other words, the redundancy index (A
introduced the concept of "bundance variation". This is a numerical value obtained by dividing the content percentage of the most duplicated clone among the clones constituting the cDNA library by the content percentage of the least duplicated clone. In reality, it is physically impossible to check the content percentage of each clone for all clones, so calculations were made for the clones obtained. There are two possible methods for calculating this overlap index. One is the specific c
In this method, colony hybridization is performed according to a conventional method using DNA as a probe, and the proportion of positive colonies in the library is measured. In this case, it is preferable to use as many types of probe cDNA as possible. The second method is to randomly pick up cDNA clones from a library, determine their nucleotide sequences, and compare the individual nucleotide sequences to estimate how many duplicate identical clones exist.

Here, using the former method, we used cD prepared as described above.
The redundancy index of the NA library was calculated.

The probes used were mouse β-actin, eIF-4A, and Vimen, which are expressed genes of neo, tk, and Ltk cells, which were added as control markers.
tin, IAP, EF-1a, ATPa
There are a total of 8 types of se-6 genes.

The results are shown in Table 1, which shows the cDNA library IJ-(S) by the conventional method shown as a comparative example and the homogenized cDNA library (EI, EI) of the present invention.
For each of I and EI), the number of positive colonies for each probe and the percentage content are shown in parentheses.

As is clear from Table 1, the cDNA library (S) obtained by the conventional method has a duplication index of more than 1,600 (estimated to be about 20,000), or the cDNA library (S) obtained by the conventional method has a duplication index of more than 1,600 (estimated to be about 20,000). 40 in the library (E DI)
decreased to Theoretically, a completely homogenized cDNA library is one with a redundancy index of 1, but if experimental errors are taken into account, the ratio of the highest content to the lowest content is 40 times seems to be a sufficient degree of uniformity.

(Effects of the Invention) As explained in detail above, the present invention enables the cloning and identification of unknown genes, the simultaneous identification of the expression patterns of multiple gene groups, and even a single set of all genes that can be expressed in organisms including humans. Provided are homogenized cDNA libraries that are widely useful in the fields of medical biological research and genetic engineering, such as the production of cDNA libraries containing cDNA libraries, and methods for producing the same.

Claims (4)

[Claims] (1) Double-stranded cDNA synthesized using mRNA as a template is denatured, and then re-associated within an arbitrary period of time, and the cDNA that has re-associated into double-strands is separated from the cDNA that remains single-stranded. 1. A method for producing a homogenized cDNA library, which comprises synthesizing single-stranded cDNA into double-stranded cDNA and cloning each cDNA. (2) The method for producing a homogenized cDNA library according to claim (1), wherein double-stranded cDNA synthesized using mRNA as a template is fragmented by providing a cohesive end in advance at its 3' end. (3) PCR the cDNA to be cloned or its fragment
A method for producing a homogenized cDNA library according to claims (1) and (2), which is amplified by a method. (4) A homogenized cDNA library produced by the method according to claims (1) to (3).