THE USE OF CLASS IIB RESTRICTION ENDONUCLEASES IN 2ND GENERATION SEQUENCING APPLICATIONS

• US 20120094847A1
• Filed: 05/05/2010
• Published: 04/19/2012
• Est. Priority Date: 05/05/2009
• Status: Active Grant

• Current Assignee: Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e)
• Original Assignee: Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e)
• Inventors: Weigel, Detlef, Warthmann, Norman
• Granted Patent: US 8,980,551 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

The present invention relates to a method for genotyping DNA molecules contained in at least one DNA sample comprising: (a) digesting the DNA molecules contained in at least one DNA sample with a class IIB restriction endonuclease to generate DNA fragments; (b) optionally separating DNA fragments comprising the recognition site for said class IIB restriction endonuclease from the remaining DNA fragments; (c) attaching at least one adaptor DNA to the 5′ and/or 3′ end of one or both strands of the DNA fragments comprising the recognition site for said class IIB restriction endonuclease obtained in a) or separated in b) to form adaptor-fragment constructs; (d) determining the sequence of at least a fraction of the DNA fragments obtained in c); and (e) assigning genotypes to said at least one DNA sample analysed based on the sequence data obtained in d). The present invention further relates to method for determining the position of DNA molecules comprised in a DNA library within the DNA sequence represented by said DNA library or within a known DNA sequence and for establishing a cross-reference between individual DNA molecules and their location in an at least three dimensional matrix.

Claims

1. A method for genotyping DNA molecules contained in at least one DNA sample comprising:

a) digesting the DNA molecules contained in at least one DNA sample with a class IIB restriction endonuclease to generate DNA fragments;

b) optionally separating DNA fragments comprising the recognition site for said class IIB restriction endonuclease from the remaining DNA fragments;

c) attaching at least one adaptor DNA to the 5′ and

/or 3′

end of one or both strands of the DNA fragments comprising the recognition site for said class IIB restriction endonuclease obtained in a) or separated in b) to form adaptor-fragment constructs;

d) determining the sequence of at least a fraction of the DNA fragments obtained in c); and

e) assigning genotypes to said at least one DNA sample analysed based on the sequence data obtained in d).

2. The method of claim 1, wherein said at least one DNA sample is a plurality of DNA samples and wherein said adaptor DNA attached in c)

(i) comprises a DNA barcode specific for each DNA sample;

or

(ii) is amplified using at least one primer binding to said adaptor DNA, wherein said primer comprises a DNA barcode specific for each DNA sample; and

wherein step d) further comprises determining the sequence of the DNA barcode specific for each sample.

3. The method of claim 1, wherein step e) comprises comparing the sequence data of said at least one DNA sample with those of a reference DNA sample and analysing for differences in the nucleic acid sequence of at least one DNA fragment of said at least one DNA sample analysed as compared to said reference DNA.

4. The method of claim 2, wherein step e) comprises comparing the sequence data of the DNA samples forming said plurality of DNA samples with each other and analysing for differences in the nucleic acid sequence of at least one DNA fragment of at least one DNA sample analysed.

6. The method of claim 1 further comprising, after step c) and prior to step d) amplifying said adaptor-fragment constructs.

7. The method of claim 1, further comprising immobilising the adaptor-fragment constructs obtained in c) on a solid surface prior to d).

10. The method of claim 7, wherein said adaptor-fragment constructs of all DNA samples are pooled prior to immobilization.

11. The method of claim 1, wherein said determining of the sequence is effected starting from both ends of the adaptor-fragment constructs.

15. The method of claim 1, wherein said attaching of the adaptor DNA is carried out using the method of attaching different DNA adaptors to the 3′

and the 5′

end of a DNA molecule comprising;

a) attaching a poly-dNTP strand terminated by ddNTP to the or each 3′

end of a single- or double-stranded DNA molecule, wherein all dNTPs are the same and wherein N in ddNTP is not N in dNTP;

b) annealing a primer comprising a 3′

poly-dNTP strand complementary to the poly-dNTP strand attached in a), wherein the N in the 3′

terminal dNTP is different from the N of the remaining poly-dNTP strand, to the nucleic acid strand of b), wherein the 5′

end of said primer is blocked;

c) removing the unpaired nucleotides of the poly-dNTP strand produced in a) and filling-in the 3′

ends of both DNA strands to form blunt ended double stranded DNA fragments;

d) adding a dNMP to the 3′

end of each DNA strand;

e) ligating to each double strand having a 3′

dNMP overhang and a 5′

phosphate a double stranded DNA adaptor having a 3′

dNMP overhang complementary to the 3′

overhang of said double strand; and

, optionally,

f) amplifying the DNA obtained in e).

16. The method of claim 1, wherein said attaching of the adaptor DNA is carried out using a Y-shaped adaptor DNA.

5. A method for determining the position of DNA molecules comprised in a DNA library relative to each other within the DNA sequence represented by said DNA library and/or within a known DNA sequence, comprising:

a) digesting DNA molecules comprised in a DNA library with a class IIB restriction endonuclease to generate DNA fragments;

b) optionally separating DNA fragments comprising the recognition site for said class IIB restriction endonuclease from the remaining DNA fragments;

c) attaching at least one adaptor DNA to the 5′ and

/or 3′

end of one or both strands of the DNA fragments comprising the recognition site for said class IIB restriction endonuclease obtained in a) or separated in b) to form adaptor-fragment constructs;

d) determining the sequence of at least a fraction of the DNA fragments obtained in c);

e) analysing for overlapping sequences between the DNA fragments sequenced in d);

f) allocating a position to said DNA fragments relative to each other and within the DNA sequence represented by said DNA library based on the sequence overlaps identified in e) and/or

g) allocating a position to said DNA fragments relative to each other and/or within a known DNA sequence.

8. A method for establishing a cross-reference between individual DNA molecules and their location in an at least three dimensional matrix, the method comprising:

a) distributing DNA molecules comprised in a library in an at least three-dimensional matrix comprising multiple distinct locations in all of said at least three dimensions such that each DNA molecule is contained in one of said locations;

b) combining the DNA molecules into different pools, such that each DNA molecule is present in at least three different pools;

c) digesting the DNA fragments contained in each pool with a class IIB restriction endonuclease to generate DNA fragments;

d) optionally separating DNA fragments comprising the recognition site for said class IIB restriction endonuclease from the remaining DNA fragments for each pool;

e) attaching at least one adaptor DNA to the 5′ and

/or 3′

end of one or both strands of the DNA fragments comprising the recognition site for said class IIB restriction endonuclease obtained in a) or separated in d) to form adaptor-fragment constructs and optionally amplifying said adaptor-fragment constructs;

wherein said adaptor DNA

(i) comprises a DNA barcode specific for each pool;

or

(ii) is amplified using at least one primer binding to said adaptor DNA, wherein said primer comprises a DNA barcode specific for each pool; and

f) determining the sequence of at least a fraction of the DNA fragments obtained in e) and the sequence of the DNA barcode attached thereto; and

g) cross-referencing all individual DNA molecules to their locations within said at least three-dimensional matrix based on the sequence data obtained in f), wherein the location of each individual DNA molecule is determined as the point of intersection of said at least three pools it is comprised in.

9. The method of claim 8, further comprising immobilising the adaptor-fragment constructs obtained in e) on a solid surface.

12. A method for attaching different DNA adaptors to the 3′

and the 5′

end of a DNA molecule comprising;

a) attaching a poly-dNTP strand terminated by ddNTP to the or each 3′

end of a single- or double-stranded DNA molecule, wherein all dNTPs are the same and wherein N in ddNTP is not N in dNTP;

b) annealing a primer comprising a 3′

poly-dNTP strand complementary to the poly-dNTP strand attached in a), wherein the N in the 3′

terminal dNTP is different from the N of the remaining poly-dNTP strand, to the nucleic acid strand of b), wherein the 5′

end of said primer is blocked;

c) removing the unpaired nucleotides of the poly-dNTP strand produced in a) and filling-in the 3′

ends of both DNA strands to form blunt ended double stranded DNA fragments;

d) adding a dNMP to the 3′

end of each DNA strand;

e) ligating to each double strand having a 3′

dNMP overhang and a 5′

phosphate a double stranded DNA adaptor having a 3′

dNMP overhang complementary to the 3′

overhang of said double strand; and

, optionally,

f) amplifying the DNA obtained in e).

13. The method of claim 12, wherein in the case that double-stranded DNA molecules are provided, these are dissociated prior to step b).

14. The method of claim 12, wherein the nucleic acid adaptor further comprises a barcode sequence.