Method of mapping DNA fragments

• US 5,667,970 A
• Filed: 05/10/1994
• Issued: 09/16/1997
• Est. Priority Date: 05/10/1994
• Status: Expired due to Fees

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Zhang, Peisen
• Primary Examiner(s): Fleisher, Mindy
• Assistant Examiner(s): MCKELVEY, TERRY ALAN
• Application Number:
  US08/240,864
• Time in Patent Office: 1,225 Days
• Field of Search: 435/6, 435/501
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6841 In situ hybridisation

Abstract

The present invention relates to a method of ordering DNA fragments in which binary overlap information, obtained by determining whether pairs of fragments hybridize to each other, is used to build a "spanning path" across a parent DNA molecule by successively attaching cloned fragments which overlap. The relative position of the many other, non-path fragments is not essential information, but can be determined. Means for eliminating false positives and false negatives is provided. The overlap relationships between fragments may be depicted as an interval graph.

Claims

1. A method of preparing a map of a parent DNA molecule, said method comprising the following steps:

(i) cleaving more than one copy of the parent DNA molecule such that different copies are cleaved at different points to generate a plurality of fragments wherein at least some of the fragments overlap;

(ii) subcloning the fragments and arranging the subclones in pairs;

(iii) performing hybridization reactions with each pair of subclones to be considered, thereby generating binary overlap data which identifies the pairs of subclones that hybridize to each other, and forming the binary overlap data into a clone-hybridization matrix;

(iv) building a first conceptual tree of the subclones from the clone-hybridization matrix obtained in step (iii), starting with any subclone as the root vertex of the first tree, by

(a) identifying a first set of the subclones which overlap the root vertex of the first tree, thereby forming a first level of the first tree;

(b) forming the next level of the first tree by identifying a next set of the subclones which overlap at least one subclone of the previous level, but where no subclone is listed in more than one level; and

(c) repeating step (iv)(b) until no further subclones can be identified which overlap at least one subclone of the previous level and have not already been listed, so that the first tree is completed and the previous level of the first tree contains a set of one or more first boundary subclones;

(v) building a second conceptual tree of the subclones from the clone-hybridization matrix obtained in step (iii) using, as the root vertex of the second tree, a first boundary subclone identified in step (iv), by

(a) identifying a first set of the subclones which overlap the root vertex of the second tree, thereby forming a first level of the second tree;

(b) forming the next level of the second tree by identifying a next set of the subclones which overlap at least one subclone of the previous level, but where no subclone is listed in more than one level; and

(c) repeating step (v)(b) until no further subclones can be identified which overlap at least one subclone of the previous level and have not already been listed, so that the second tree is completed and the previous level of the second tree contains a set of one or more second boundary subclones;

(vi) attempting to form a spanning path, where said spanning path extends from a second boundary subclone identified in step (v), through a series of consecutive subclones from successive levels of the second tree, wherein each consecutive subclone overlaps both the subclone which precedes it as well as the subclone that follows it in the series, to the first boundary subclone, and wherein the spanning path cannot thus be formed;

(vii) identifying the subclone in the second tree which cross-hybridizes to the greatest number of the other subclones in the second tree;

(viii) eliminating the subclone identified in step (vii) from the second tree;

(ix) attempting to form the spanning path, if the spanning path cannot be formed, then steps (vii) to (ix) are repeated until the spanning path can be formed; and

(x) forming the spanning path into a map of the parent DNA molecule.

2. The method according to claim 1, wherein each hybridization reaction is performed by hybridizing both subclones in the pair with a probe which is produced by transcription from a T3 promoter at the end region of one of the subclones in the pair.

3. The method according to claim 1, wherein each hybridization reaction is performed by hybridizing both subclones in the pair with a probe which is produced by transcription from a T7 promoter at the end region of one of the subclones in the pair.

4. The method according to claim 1, further comprising eliminating from the clone-hybridization matrix, prior to step (iv), pairs of subclones which do not exhibit reciprocal hybridization.

5. The method according to claim 2, further comprising eliminating from the clone-hybridization matrix, prior to step (iv), pairs of subclones which do not exhibit reciprocal hybridization.

6. The method according to claim 3, further comprising eliminating from the clone-hybridization matrix, prior to step (iv), pairs of subclones which do not exhibit reciprocal hybridization.

7. The method according to claim 1, further comprising making a graph of the locations of the subclones in the map by the steps comprising reordering the clone-hybridization matrix by means of a weighted distance scheme, said weighted distance scheme comprising assigning each subclone in the spanning path a weight which is equal to the order of the subclone in the spanning path, and forming the reordered clone-hybridization matrix into a graph.

8. The method according to claim 2, further comprising making a graph of the locations of the subclones in the map by the steps comprising reordering the clone-hybridization matrix by means of a weighted distance scheme, said weighted distance scheme comprising assigning each subclone in the spanning path a weight which is equal to the order of the subclone in the spanning path, and forming the reordered clone-hybridization matrix into a graph.

9. The method according to claim 3, further comprising making a graph of the locations of the subclones in the map by the steps comprising reordering the clone-hybridization matrix by means of a weighted distance scheme, said weighted distance scheme comprising assigning each subclone in the spanning path a weight which is equal to the order of the subclone in the spanning path, and forming the reordered clone-hybridization matrix into a graph.

10. The method according to claim 4, further comprising making a graph of the locations of the subclones in the map by the steps comprising reordering the clone-hybridization matrix by means of a weighted distance scheme, said weighted distance scheme comprising assigning each subclone in the spanning path a weight which is equal to the order of the subclone in the spanning path, and forming the reordered clone-hybridization matrix into a graph.

11. The method according to claim 5, further comprising making a graph of the locations of the subclones in the map by the steps comprising reordering the clone-hybridization matrix by means of a weighted distance scheme, said weighted distance scheme comprising assigning each subclone in the spanning path a weight which is equal to the order of the subclone in the spanning path, and forming the reordered clone-hybridization matrix into a graph.

12. The method according to claim 6, further comprising making a graph of the locations of the subclones in the map by the steps comprising reordering the clone-hybridization matrix by means of a weighted distance scheme, said weighted distance scheme comprising assigning each subclone in the spanning path a weight which is equal to the order of the subclone in the spanning path, and forming the reordered clone-hybridization matrix into a graph.