RU2625012C2 - Method for preparation of genomic libraries of limited selections of locuses from degraded dna - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method involves hydrolysis of endonuclease restriction genomic DNA forming fragments with a 5'-sticky end and partial end-blunting by a polymerase reaction in the presence of an incomplete set of deoxyribonucleoside triphosphates. Ligation of fragments with adapters having a sticky end, complementary to the sticky end of a target fragment modified by partial blunting, selection of target fragments together with fragments selection by length and amplification with primers, complementary to the adapters. Sequential hydrolysis of genomic DNA can be carried out by two restriction endonucleases that form various sticky ends of fragments, first the endonuclease restriction sensitive to nucleotides chemical modification, then its isoschizomer insensitive to modification. During library fragments amplification, primers are used that include: a site complementary to the adapters, a site complementary to the rest of the restriction endonuclease recognition site, and a site of a specific 1-4 nucleotide extension at the 3' end of the primer to limit the selection of library loci.

EFFECT: invention allows to obtain genomic libraries of degraded DNA samples in a short time and with high selectivity with respect to the target fragments.

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Description

The present invention relates to the field of biotechnology, in particular to molecular biology, and can be used in genetic studies that require the preparation of genomic libraries of limited samples of loci from degraded DNA, regardless of the biological origin of the studied genomes and regardless of the studied classes of nucleotide sequences (e.g. regulatory areas of genes, repetitive elements of the genome, etc.), including retrospective genetic studies in the field of oncol ohii.

A method is known from the prior art that lists reagents and methods for preparing a genomic library with a limited selection of loci from bisulfite-converted genomic DNA by hybridizing a sample with locus-specific probes (WO 2015014759 A1, 02/05/2015).

The disadvantages of this method are: the duration of the protocol; obtaining a large number of fragments that are not related to the target loci of the genome due to nonspecific hybridization of DNA fragments with reduced, due to the processing of sodium bisulfite, informativeness of nucleotide sequences.

The target genome loci (target DNA fragments) are the sections of genomic DNA, the properties of which should be studied in accordance with the purpose of a specific study.

Another known method is the selective amplification of target loci of high multiplex PCR using specific primers (WO 2014018080 A1, 01/30/2014).

The disadvantages of this method are: an increase in the proportion of fragments not related to the target loci, with an increase in the number of studied loci due to the formation of non-specific PCR products; the inability to detect chemical modifications of the nucleotides (for example, DNA methylation).

Also known is a method consisting in processing a sample of genomic DNA with a restriction endonuclease with a specific recognition site, followed by adapter-mediated amplification of the obtained fragments with a primer with a specific 3'-extension cord (RU 2472859 C1, 01/20/2013).

The disadvantages of this method are: high demands on the quality of the DNA of the studied samples, which makes it impossible to use this method for retrospective genetic studies, since the DNA from available tissue samples stored in paraffin blocks for histological studies is highly fragmented.

A known method in which when working with DNA from paraffin blocks using preliminary selection of DNA fragments longer than 500 bp (N. Gu, C. Bock, et al. Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution // 2010. Nat Methods, 7 (2), 133-136).

The disadvantages of this method are: the presence in the resulting libraries of a high proportion of fragments that are not related to the studied sample of loci; low quantitative yield of libraries. The applicability of the method is limited to a small part of fresh paraffin blocks, from which it is possible to isolate DNA fragments longer than 500 bp.

A method is also known in which the method of sequencing a genomic library of limited samples of loci converted with sodium bisulfite was applied to determine the methylation status of individual CpG pairs in CpG-rich regions of the human genome. This method includes the following steps: treatment of genomic DNA with restriction enzyme MspI, complete blunting of the ends of the fragments to prevent self-ligation of the fragments to obtain a sticky 3'-A end, ligation with adapters with a sticky 3'-T end, selection of fragments in length, processing of the fragments with sodium bisulfite, amplification of library fragments (N. Gu, ZD Smith et al. Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling // 2011. Nat Protocols, 6 (4), 468-481).

The disadvantage of this method is the method for the complete blunting of the ends of the fragments used to prevent self-ligation, which makes it impractical to prepare libraries from samples of degraded DNA. With this method of blunting, short fragments initially present in a sample of degraded DNA cannot be separated from fragments obtained by treatment with a restriction endonuclease with a specific recognition site at the stage of selection of fragments in length, and fall into the genomic library, forming a significant fraction of non-targeted fragments (figure one).

The objective of the invention is a method for preparing genomic libraries of limited samples of loci from degraded DNA, which allows to reduce the proportion of DNA fragments that are not related to the target parts of the genome, which is achieved by forming a difference between the ends of the target fragments and fragments formed as a result of DNA degradation using the proposed methods for modifying the ends fragments.

The problem is solved by the method consisting in the fact that hydrolysis of genomic DNA is carried out with a restriction endonuclease that forms fragments with a 5'-sticky end; partial blunting of the ends by polymerase reaction in the presence of an incomplete set of deoxyribonucleoside triphosphates, ligation of fragments with adapters with a sticky end, complementary to the modified sticky end of the target fragment, selection of target fragments together by selection along the length of the fragments and amplification with primers, complementary adapters.

The composition of a limited sample of loci is determined by the nucleotide composition of the recognition site used for the hydrolysis of restriction endonuclease DNA and the selection of fragments along the length in accordance with the design of a particular study. The maximum length of DNA fragments of a limited sample of loci is determined by the technical capabilities of the technology for its subsequent analysis, for example, the maximum allowable DNA fragment length by sequencing. The method can be used to study the genomic DNA of any kind of organisms, provided that in its sequence there are recognition sites of the selected restriction endonuclease located at a distance not exceeding the maximum length of DNA fragments for which subsequent analysis is available.

Hydrolysis of genomic DNA can be carried out sensitive to chemical modification of nucleotides by a restriction endonuclease and its insensitive to modification by an isoshizomer, forming various sticky ends of fragments.

For amplification, primers can be used that contain a site complementary to the adapters, a site complementary to the rest of the restriction endonuclease recognition site, a specific extension site of 1-4 nucleotides at the 3 'end of the primer, to limit the selection of library loci.

The method is practically carried out as follows.

Stages of the method: (1) site-specific hydrolysis of DNA; (2) modification of the ends of DNA fragments by partial blunting; (3) site-specific ligation of DNA fragments with adapters; (4) selection of DNA fragments along the length; (5) amplification of DNA fragments.

Genomic DNA is treated with a restriction endonuclease with a recognition site characteristic of the researcher of interest in the genome sequence class, which forms fragments with a 5'-sticky end. In a restriction mixture with a total volume of 11.4 μl, 0.1-10 μg of native DNA, 1.1 μl of the corresponding 10-fold buffer and 20 units of the selected restriction endonuclease are added. The mixture is incubated at a temperature optimal for the selected enzyme for 16 hours.

The use of restriction endonucleases for DNA hydrolysis that are sensitive to chemical modifications of nucleotides and their insensitive to such modifications of isoshizomers makes it possible to identify epigenetic chemical modifications of nucleotides by the presence or absence of fragments of genomic loci in the resulting libraries. In this case, DNA hydrolysis is carried out sequentially, first with a restriction endonuclease that is sensitive to chemical modifications of the nucleotides, and then it is insensitive to such modifications with the isoshizomer.

To modify the ends of the fragments, which prevents self-ligation of the fragments and forms the difference between the ends of the target fragments and the ends of the fragments formed as a result of DNA degradation, the hydrolysis products are treated with 2.5 u.a. mutant recombinant of E. coli polymerase I lacking 3'-5'-exonuclease (corrective) activity (Klenov exo fragment) for 30 min at 37 ° C, after which it is necessary to inactivate the incubation enzyme for 10 min at 75 ° C . Partial blunting in the presence of 0.5 μl of an incomplete set of deoxyribonucleotide triphosphates (each 1 mM each) simultaneously prevents fragments self-ligation and forms a site-specific end for fragments obtained by restriction endonuclease hydrolysis, which differs from the end of fragments resulting from DNA degradation. If the restriction enzyme buffer is incompatible with the Klenov exo fragment buffer, it is necessary to purify DNA fragments, for example, using the Agencourt AMPure XP reagent (Beckman Coulter Inc.) according to the manufacturer's protocol, or, if possible, correct the buffer to the required molar composition.

Site-specific ligation of fragments with adapters with a sticky end complementary to the sticky end of the fragment obtained by partially blunting the ends of the fragments formed by the restriction endonuclease. The design of the 5'-sticky end of the adapter must be carried out taking into account the recognition site of the restriction endonuclease used to prepare the library and the method used to blunt the ends of the fragments. An example of such a design is shown in Figure 2. 0.8 μl of each of the 15 μM adapters are added to the sample; 1.8 μl dATP 10 mM; 1.6 μl of ligase 200,000 CEU / ml. Since the ligase buffer is compatible with the buffer of the Klenov exo fragment, it is sufficient to correct the reaction buffer by adding 0.6 μl of a ten-fold ligase buffer. Adapters can be prepared by annealing from synthetic oligonucleotides, for which an equimolar mixture of oligonucleotides is heated to 95 ° C and cooled at a rate of 2 ° / min. If, to prevent the formation of adapter concatemers, adapters were used that were not cinned at the 5'-end from the ligated 5'-sticky end, then nick translation should be performed using 1 unit a. polymerase with the addition of 1 μl of a mixture of all deoxyribonucleotides (dNTP) of 2.5 mm each. Adapters compatible with various instruments for high-performance parallel sequencing of fragments can be used, for example, Roche / 454 Life Sciences, Applied Biosystems / SOLiD, Ion Torrent / Proton, Illumina, suitable performance according to the manufacturer's protocol. The present invention does not impose requirements on the structure of the main part of the adapter. Of fundamental importance is the design of the 5'-sticky end of the adapter, which must be carried out taking into account the recognition site of the restriction endonuclease used to prepare the library, and the method used to partially blunt the ends of the fragments. An example of such a design is shown in figure 2.

Selection of library fragments to reduce the selection of loci can be performed by selection of fragments along the length, for example, in a PippinPrep instrument (Sage Science, USA) according to the manufacturer's protocol, or by electrophoresis of library fragments in a 2% agarose gel, in the presence of molecular weight fragments in neighboring tracks. The area of the gel containing the library fragments of the length researcher of interest is excised with a disposable scalpel, after which the fragments are isolated from the gel, for example, using the GeneJET Gel Extraction Kit (ThermoFisher scientific) reagent kit for DNA extraction from agarose gel. Before gel electrophoresis, the sample was purified from DNA-binding proteins and after electrophoresis, the sample was purified from electrophoretic buffer, for example, using the Agencourt AMPure XP reagent (Beckman Coulter Inc.) according to the manufacturer's protocol.

The amplification of the library with primers complementary to the adapters is carried out under conditions optimal for the primers complementary to the adapters used, depending on the platform selected for clonal sequencing of the library fragments according to the manufacturer's protocol. When using primers containing a portion complementary to the adapters, a portion complementary to the rest of the restriction endonuclease recognition site, and a portion of a specific extension of 1-4 nucleotides at the 3 'end of the primer, it is possible to reduce the size of the resulting genomic library. The combination of a restriction endonuclease recognition site and a specific primer extension allows the selection of genomic loci to form a library of fragments belonging to different classes of genome sequences, for example, CpG islands, repeating families, etc.

Only fragments subsidized on both sides by adapters can participate in the amplification process, which is possible only if the fragment on both sides had ends corresponding to the ends formed by blunting the 5'-sticky end obtained by treating genomic DNA with a selected restriction endonuclease. Thus, the selection of target fragments of the library occurs, since DNA fragments formed as a result of DNA degradation are very unlikely to have ends suitable for ligation of adapters.

The figure 1 shows the differences in the content in the prepared genomic library of non-target fragments in the preparation of genomic libraries using: high molecular weight DNA; fragmented DNA according to previously proposed protocols; fragmented DNA in accordance with the present invention. Thick lines indicate the target loci of the genome, thin lines indicate non-target loci of the genome, the inclusion of which in the library is undesirable. Adapters are indicated by thick gray lines. The ends of the fragments formed as a result of DNA hydrolysis by restriction endonuclease are indicated by corners. The ends of the fragments formed as a result of DNA degradation are free. The ends of fragments prepared for ligation with adapters are indicated by semicircles. In the case of preparing a genomic library from fragmented DNA according to previously proposed protocols, PCR with primers complementary to the adapters leads to the formation of a significant number of non-target elements of the genomic library.

Figure 2 shows an example of site-specific ligation of adapters with 5'-sticky ends of fragments, partially stupid in the presence of an incomplete set of deoxyribonucleotide triphosphates: as a result of hydrolysis of genomic DNA with a restriction endonuclease (the Ah1I restriction enzyme recognition site is considered in the example) fragments with a 5'-sticky palindromic end; as a result of partial blunting of the ends of the fragments to prevent self-ligation of the library fragments and adapters (polymerase reaction with an incomplete set of deoxyribonucleotide triphosphates, in the example dCTP and dTTP), fragments with a 5'-sticky non-palindromic end corresponding to a part of the restriction endonuclease recognition site are formed; ligation is carried out with an adapter having a 5'-sticky end complementary to the resulting 5'-sticky end of the fragment.

The defining distinguishing features of the proposed method, in comparison with analogues, are simultaneously: high selectivity for target fragments, short preparation of genomic libraries, the ability to work with samples of degraded DNA, for example, from paraffin blocks.

Claims (1)

A method for preparing genomic libraries of limited samples of loci from degraded DNA, limited by the selection of DNA fragments along the length of the DNA fragment after treatment with a restriction endonuclease, characterized in that the genomic DNA is hydrolyzed with a restriction endonuclease, forming fragments with a 5'-sticky end, partial blunting of the ends by polymerase reaction in the presence of an incomplete set of deoxyribonucleoside triphosphates, ligation of fragments with adapters with a sticky end complementary to modified partial blunting at the end of the target fragment, selection of the target fragments together by selection along the length of the fragments and amplification with primers complementary to the adapters, and the sequential hydrolysis of genomic DNA can be carried out by two restriction endonucleases that form the various sticky ends of the fragments, first sensitive to the chemical modification of nucleotides with a restriction endonuclease, then insensitive to modification by the isoshizomer, and when amplifying fragments of the library, primers are used that contain: ary adapters region complementary residue restriction endonuclease recognition site, and a site-specific extension length 1-4 nucleotides at the 3 'end of the primer for sampling limitations loci library.

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