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Capturing sequences adjacent to type-IIS restriction sites for genomic library mapping

Inactive Publication Date: 2006-10-05
AFFYMETRIX INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0019] The present invention provides methods for identifying specific oligonucleotide sequences using Type-IIs endonucleases in sequential order to capture the ambiguous sequences adjacent to the Type-IIs recognition sites. These ambiguous sequences can then be probed sequentially with probes specific for the various combinations of possib

Problems solved by technology

Although each of these methods is capable of generating useful physical maps of genomic DNA, they each involve complex series of reaction steps including multiple independent synthesis, labelling and detection procedures.
Further, these endonucleases generally cleave outside of their recognition site, thus producing overhangs of ambiguous base pairs.
However, this method can be time consuming and yields only the sequence of the overhang region.
Others have proposed the use of large numbers of oligonucleotide probes to provide the complete nucleic acid sequence of a target nucleic acid but failed to provide an enabling method for using arrays of immobilized probes for this purpose.
These methods, while providing useful mapping information, are very time consuming when applied to very large genome fragments or other nucleic acids.

Method used

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  • Capturing sequences adjacent to type-IIS restriction sites for genomic library mapping
  • Capturing sequences adjacent to type-IIS restriction sites for genomic library mapping
  • Capturing sequences adjacent to type-IIS restriction sites for genomic library mapping

Examples

Experimental program
Comparison scheme
Effect test

example 1

1. Digesting High Molecular Weight DNA with EarI

[0084] 4 μg of λ DNA was treated with 4 units of EarI in 10 μl at 37° C. for 4 hours. The reaction was then heated to 70° C. for 10 minutes. Cleavage was verified by running 5 μl of the sample on an agarose gel to determine complete cleavage. The remaining 5 μl was brought to 40 μl (final concentration of 50 ng / μl λ DNA).

2. Klenow Fill-In Reaction

[0085] 4 μl of the digested λ DNA was added to 0.5 μl of 10× Klenow Buffer, 0.5 μl 2 mM dNTPs, and 0.05 μl of 0.25 units of Klenow fragment. The reaction mixture was incubated for 20 minutes at 25° C., followed by 10 minutes at 75° C. Similar results were also obtained using T4 DNA polymerase for the fill-in reaction.

3. Preparing Adapter Sequences

[0086] Two separate adapter sequences were prepared, adapter sequence 1 and adapter sequence 2. Adapter sequence 1 is used in the first ligation reaction whereas adapter 2 is used for the second. As each adapter and its ligation are somewhat d...

example 2

[0104] The above capture methods were applied to a genomic library of 12 known cosmids from yeast chromosome IV. The clones have been previously physically mapped using EcoRI-HindIII fragmentation. The specific library, including known zap positions and overlap of the 12 cosmids, is illustrated in FIG. 6.

[0105] The twelve genomic clones were constructed in a pHC79 vector, in E. coli host HB101. Cosmid DNA was prepared from 3 ml cultures by an alkaline lysis miniprep method. The miniprep DNA was digested with EcoRI and HindIII to confirm the known fingerprint of the large cloned inserts. Cosmid DNA was treated with linear DNAase, Plasmid-Safe™ DNAse, at 37° C. for 15 minutes, followed by heat inactivation. The DNAse treatment was carried out to remove any potential spurious EarI digested sites resulting from contaminating bacterial DNA. This leaves cosmid DNA substantially untouched. After confirming the presence of clean banding cosmid DNA, the resulting cosmids were then subjected...

example 3

Simulated Annealing

[0109] The correlation scores from yeast chromosome IV, above, were used to construct a best fitting contig, using the simulated annealing process as described by Cuticchia, et al., The use of simulated annealing in chromosome reconstruction experiments based on binary scoring, Genetics (1992) 132:591-601. A global maximum was sought for the sum of correlation coefficient scores for a given sequence of cosmids in the randomly constructed and permutated contig. The resulting high scoring contigs for all 12 cosmids and for the 10 “strong-signal” cosmids are shown below. Each cosmid was assigned a rank based upon the known position of that cosmid, and these are as follows:

TABLE 1Cosmid NumberCosmid Rank9371A8552B808719481298583958348024582536950979460880649983110 

[0110] Simulated annealing of all twelve cosmids produced the following ordering:

(1 2 3 4) (7 6 5) A B (8 9 10)

[0111] Inclusion of the weaker signal cosmids, A and B, results in some shuffling of the pr...

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Abstract

The present invention relates to novel methods for sequencing and mapping genetic markers in polynucleotide sequences using Type-IIs restriction endonucleases. The methods herein described result in the “capturing” and determination of specific oligonucleotide sequences located adjacent to Type-IIs restriction sites. The resulting sequences are useful as effective markers for use in genetic napping, screening and manipulation.

Description

[0001] This application is a continuation-in-part of U.S. application Ser. No. 08 / 307,881, filed Sep. 16, 1994, which is hereby incorporated by reference in its entirety for all purposes.[0002] Research leading to the present invention was funded in part by NIH grant Nos. 5-F32-HG00105 and RO1 HG00813-02, and the government may have certain rights to the invention.[0003] The present invention generally relates to novel methods for isolating, characterizing and mapping genetic markers in polynucleotide sequences. More particularly, the present invention provides methods for mapping genetic material using Type-IIs restriction endonucleases. The methods herein described result in the “capturing” and determination of specific oligonucleotide sequences located adjacent to Type-IIs restriction sites. The resulting sequences are useful as effective markers for use in genetic mapping, screening and manipulation. BACKGROUND OF THE INVENTION [0004] The relationship between structure and funct...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12P19/34
CPCC12Q1/6806C12Q1/6809C12Q1/6827C12Q1/683C12Q1/6855C12Q1/6869C12Q2600/156C12Q2565/501C12Q1/6874C12Q2525/191C12Q2521/313C12Q2525/179C12Q2525/161C12Q2525/155C12Q2525/143C12Q2525/131
Inventor SAPOLSKY, RONALDLIPSHUTZ, ROBERTGINGERAS, THOMAS
Owner AFFYMETRIX INC
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