WO2001006014A1

WO2001006014A1 - Removing repetitive sequences from dna

Info

Publication number: WO2001006014A1
Application number: PCT/US2000/019449
Authority: WO
Inventors: Xin-Yuan Guan
Original assignee: American Laboratory Technologies, Inc.
Priority date: 1999-07-19
Filing date: 2000-07-18
Publication date: 2001-01-25
Also published as: AU6350800A

Abstract

Methods for removing repetitive sequences from a nucleic acid probe are provided. The methods comprise steps of providing a nucleic acid probe containing repetitive sequences, providing human DNA containing repetitive sequences that hybridize with the repetitive sequences of the probe, hybridizing the probe and the human DNA, removing the hybridized repetitive sequences and recovering the remainder of the probe.

Description

REMOVING REPETITIVE SEQUENCES FROM DNA This application claims priority based on Provisional Application Serial No. 60/128,307, filed April 8, 1999.

FIELD OF THE INVENTION

The present invention relates generally to the study of cytogenetics . More particularly, it concerns improvements in techniques for analyzing chromosome abnormalities.

BACKGROUND OF THE INVENTION

It has been known for decades that chromosome rearrangements exist in most if not all human tumors (Miteiman et al., "Human Gene Mapping," Cytogenetic Cell Genet., 58:653- 79 (1991)) and certain human hereditary diseases (Frezal et al., "Human Gene Mapping 11," Cytogenet Cell Genet., 58:986- 1052 (1991)). Distinct chromosomal abnormalities in tumors lead to the activation of proto oncogene products, creation of tumor-specific fusion proteins, or inactivation of tumor suppresser genes. Since chromosome banding techniques were developed, cytogenetic study of nonrandom chromosome abnormalities in malignant cells has become an integral part of the diagnostic and prognostic work-up of many human cancers (Sandberg, 1990; Trent et al . , 1990). Additionally, cytogenetic studies followed by molecular analysis of recurring chromosomal rearrangements have greatly facilitated the identification of genes related to the pathogenesis of both hereditary diseases and cancer. For example, the tumor suppresser gene Rb was identified based on the observation of deletion of chromosome 13ql4 in retinoblastoma (Yunis and Ramsay, 1978) and the proto-oncogene c-myc was shown to be involved in the chromosome translocation t(8;14) in human Burkett's lymphomas (Zech, et al., 1976).

However, not all cytogenetically visible chromosome rearrangements (e. g. , complex chromosome rearrangements, small ring chromosomes, and unidentifiable de novo unbalanced translocation) can be determined by conventional cytogenetic banding analysis. This technique limitation prevents complete karyotypic analysis in many human cancers, particularly solid tumors. This technical limitation has been countered by the development of the fluorescence in situ hybridization (FISH) technique (Pinkel, et al . , "Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4," Proc. Natl. Acad. Sci. USA 85:9138-42 (1988)). After a decade of effort, a variety of fluorescent painting probes including human whole chromosome painting probes (WCPs) (Guan, et al., "Rapid generation of whole chromosome painting probes (WCPs) by chromosome microdissection," Genomics, July 1; 22 (1) : 101-107 (1994)), chromosome arm painting probes (CAPs) (Guan, et al., "Rapid generation of human chromosome arm painting probes (CAPs) by chromosome microdissection," Nature Genet 12:10-11 (1996)), and chromosome band-specific painting probes (Guan, et al., "DNA sequence amplification in human prostate cancer identified by chromosome microdissection: potential prognostic implications," Clinic Cancer Res., 1:11-18 (1995)), have been developed and widely applied to both research and clinical diagnosis .

A major problem with currently available fluorescent painting probes and other genomic DNA probes, such as yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) containing human genomic DNA fragments, is the background signal caused by the cross-hybridization of repetitive sequences existing in the probes. Human genomic DNA contains many different kinds of repetitive sequences. Some of them, such as the short highly repeat sequences Alu and the long repeat sequence LI, will appear in genomic DNA approximately every few kilo-bases. One solution has been to block these repetitive sequences during hybridization. Conventional blocking methods have been used in which total human genomic DNA for commercially available human Cot-1 DNA containing several different repetitive sequences are applied to pre-hybridize with the repetitive sequence in the probe. However, disadvantages of conventional background blocking methods include: 1) the pre-hybridization process tends to decrease the fluorescent signals due to self-hybridization of the unique sequence in the probe before hybridization to the target sequences; 2) experience is required to effectively block the repetitive sequences in the probe; 3) human Cot-1 is expensive; and 4) the pre-hybridization process is time- consuming .

Under a license agreement with NIH, American Laboratory Technologies, Inc. of Arlington, Virginia, manufactures and commercially markets microdissected probes generated at Dr. Jeffrey Trent's laboratory at the National Human Genome Research Institute, NIH. Substantial time and effort were expended to determine the optical ratio of Cot-1 DNA to each microdissected painting probe during commercial preparation. The problem was exacerbated preparing probes for multi-color FISH, Fast-FISH and interphase FISH, since these require higher quality painting probes with less noise.

As a consequence, it is a prime object of the present invention to provide a method of rendering ineffective the repetitive sequences inherent in chromosome analysis so that these sequences, which are basically irrelevant to a determination of chromosomal abnormalities, do not present a background signal or noise that interferes with DNA analysis to determine chromosomal abnormalities.

It is a further object of my invention to render such background noise ineffective in a relatively economic and simple manner when compared to the repetitive sequence blocking techniques presently in use.

It is still another object of the present invention to provide for the generation of repetitive sequence-depleted libraries that will meet the demands of advanced FISH technologies, such as FAST FISH and MULTI-COLOR FISH, decrease the costs of manufacture of such probes, and simplify the protocols for using these probes in FISH. Thus, the probes prepared using the techniques according to the present invention will be of higher quality than those available commercially, and permit significantly faster and more consistent results.

SUMMARY OF THE INVENTION

In accordance with the present invention, a process is employed by means of which undesirable repetitive sequences are removed from a nucleic acid probe rather than merely being blocked when the probe is hybridized with the target DNA. The invention is also directed to the unique products that are formed after such repetitive sequences have been removed from the probe.

Thus, stated broadly, my invention comprises (a) providing a nucleic acid probe containing repetitive sequences that detract from the effectiveness of the probe. Also provided is (b) human DNA containing repetitive sequences that hybridize with the repetitive sequences of the probe. In step

(c) the human DNA and the probe are reacted so that the undesirable repetitive sequences of the probe and the human DNA hybridize. Thereafter, (d) the hybridized repetitive sequences are removed from the mixture, and (e) the remaining portions of the probe are recovered from the mixture in the form of a nucleic acid probe having a substantial portion of the repetitive sequences removed therefrom.

In somewhat more restricted form, my invention specifies in the process above-described that the human DNA that is part of the mixture of step (c) is amplified, microdissected DNA fragments. Further, said fragments are labeled with biotin, preferably by nick translation.

With respect to the reaction that takes place in step

(c) , the reaction is a hybridization between the microdissected DNA fragments and the probe that has biotin- labeled, repetitive sequences. After the reaction has been completed, the hybridized repetitive sequences are removed in step (d) by incubating the product of step (c) with avidin and then subtracting the hybridized repetitive sequences with phenol. Such hybridized sequences remain in solution after the addition of a salt of a weak acid, e . g. , sodium acetate, and the probe is recovered as a precipitate using a PCR primer .

In another aspect of this invention that is directed to the use of the improved probe that has its repetitive sequences removed, the above-described process is performed. Thereafter target chromosomal DNA containing repetitive sequences that hybridize with the repetitive sequences formerly in the probe are provided. However, when the improved probe according to the present invention is applied to the target chromosomal DNA, the repetitive sequences of the target DNA will not have complementary repetitive sequences of the probe with which to bind. Thus, the hybridization of the probe and target chromosomal DNA will not be confused by hybridization of undesirable repetitive sequences, and genomal rearrangements in the target DNA will be more easily identified.

These and other objects, features and advantages of my invention will be more readily ascertained by reference to the following, detailed description of a specific embodiment of my invention, which is directed to actual production of probes according to my invention and is not meant as a limitation to the scope of that invention.

DETAILED DESCRIPTION OF THE INVENTION

In the following detailed process as carried out in the laboratory, all painting probes were obtained from the laboratory of Dr. Jeffrey Trent at the National Genome Research Institute, National Institutes of Health (NIH) , Bethesda, Maryland. The process took place by the hereinafter identified steps in the order in which they are described.

1. Microdissection and PCR Amplification

Five copies of microdissected DNA fragments from target chromosome were pooled 5ul collection solution containing pepsin. The dissected DNA was incubated at 37°C. for one hour and then directly amplified with UNI (5' CGGGAGATCCGACTCGAGNNNNNNATGTGG) by PCR.

2. Preparation of Unique Primer PCR Products

Since UNI primer contains a random hexamer which may amplify any existing DNA, the degenerate primer (UNI) of microdissected DNA fragments is replaced with a unique sequence primer (RLl) in order to specifically recover the

selected microdissected DNA fragments. 2μl UNI of amplified,

dissected DNA products were amplified by PCR with UN2 primer which shares 22 bases at the 3' -end with the UNI (5' CGGGAGATCCGACTCGAGNNNNNNATGTGG-3') • The reaction was cycled 5 times at 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min,

with the final extension at 72 °C for 5 minutes. Finally, 2μl of UN2 amplified products were amplified with RLl which shares 18 bases at the 5' -end with UN2 (5' -CTCGGGAGATCTGACTCGAG-3' ) . The reaction was cycled 20 times at 94° for 1 min, 56° for 1 min, and 72° for 1 min, with the final extension at 72° for 5 minutes .

3. Preparation of Biotin-Labeled Human Repetitive Sequences and Hybridization

1) 18 human DNA fragments (1.4-8 kb) containing various repetitive sequences were cloned into plasmid, which were then amplified and purified. lOOug DNA were labeled with biotin by nick translation.

2) lOOng microdissected DNA (RL-1) was hybridized with

10 ug of biotin-labeled human repetitive sequences in 20 μl

hybridization solution. (6X SSC, 0.2% SDS) at 55 °C overnight.

4. Subtraction of Repetitive Sequences from Microdissected DNA

1) After hybridization, 20 ul Avidin (5 ug/ml) was added and incubated at 37 °C for 30 min.

2) 60 ul ddH20 and then 100 ul Phenol were added. They were mixed by votex for 30 seconds and centrifuged at 14,000 rpm for 5 min. 3) The supernatant was transferred to a clean tube and 100 ul chloroform added. They were mixed by votex for 10 seconds and centrifuged at 14,000 rpm for 3 min.

4) The supernatent was transferred to a clean tube, and 1/10 3M NaAc and 2.5 volume 100% EtOH added. They were well mixed and precipitated at -20 °C overnight.

5) Centrifugation at 14,000 rmp for 30 min was supplied. The solution was carefully separated and the

precipitate air-dried. 10 μl dH20 was added.

5. Recovery of a Microdissected DNA by PCR.

After subtraction, microdissected DNA fragments containing unique sequences were recovered by PCR using RL-1 primer. The PCR reaction was cycled 30 times at 94°C for 1 min, 60 °C for 1 min, and 72 °C for 1 min, with the final extension at 72 °C for 5 min. The PCR result was then checked

by running 5 μl PCR products on 1% agarose gel .

The subtraction was repeated until most of the repetitive sequences were removed. The results were checked with FISH.

Microclone libraries were generated in the following manner: 1. PCR Product Purity

Recovered microdissected unique sequence PCR products were analyzed on an agarose gel and purified using the Wizard^® PCR Preps DNA Purification System (Promega, Madison, Wisconsin) .

2. Microcloning

To 1-2 μl of purified PCR fragments were added 1 μl of

Tag DNA polymerase reaction 10X Buffer and 1 μl of 25 mM

MgCly:dATP was added to a final cone. Of 0.2 mM. 5 units of Tag DNA Polymerase was then added. Deionixed water to a final

reaction volume of 10 μl was supplied, and the mix incubated

at 70°C for 15-30 minutes. 1-2 μl was used in a ligation

reaction with one of Promega' s pGEM^®-T vectors. (Promega, Madison, Wisconsin) . 25 ng PCR product was added to a ligation in which 50 ng of 3.0 Kb vector was used as a 3:1 insert: vector molar is desired. Successful cloning of an insert in the pGEM^® T Vectors interrupts the coding sequence

of 7-galactosidase; recombinant clones can be identified by

color screening on indicator plates.

3. Transformations Using the pGEM^® T Vectors Ligation Reactions

JM109 High Efficiency Competent Cells were used for transformations. Briefly, we prepared 2 LB/ampicillin/IPTG/X-

Gal plates for each ligation reaction and added 2 μl of each

ligation reaction to 50 μl of cells and stood on ice for 20 min, followed by heat shock at 42°C for 45-50 seconds in a water bath. 950 μl of SOC medium were added and the mix

incubated 1.5 hours at 37°C with shaking and then plated.

The following criteria were used to evaluate the quality and specificity of the library' s chromosome arm painting probes;

1. A strong and distinct fluorescence signal should be detected by each chromosome arm library painting probe. Cot-1 DNA and other block procedure will be not needed. Biotin and avidin labeling methods are used for labeling arm probes .

2. The fluorescence signal along the painting area of chromosome arm should be homogeneous and there should be no gaps in the painting area.

3. Without Cot-1 and other blocked DNA, each chromosome arm painting probe should be specific for one pair of homologous chromosome arms and should not hybridize to other chromosome areas.

4. The advantage of repetitive sequence-depleted libraries of microdissected chromosome arm painting probes was tested and evaluated. New libraries' probes make significant progress for Fast-FISH and make things much easier to be controlled for making multi-color FISH probes. Also, the libraries are a new source to make interphase FISH probes. While my invention has been described with reference to the foregoing best mode of carrying out my invention, it will be apparent to those of skill in this art that modifications and additions can be made to that detailed embodiment without departing from the spirit of the invention. As to all such modifications and additions, it is desired that they be included within the purview of my invention, which is to be limited only by the scope, including equivalents, of the following appended claims .

Claims

I CLAIM:

1. A method of removing repetitive sequences from a nucleic acid probe, comprising:

(a) providing a nucleic acid probe containing repetitive sequences that detract from the effectiveness of the probe;

(b) providing human DNA containing repetitive sequences that hybridize with the repetitive sequence of the probe;

(c) reacting a mixture of the human DNA and the probe so that the repetitive sequences of the probe and the human DNA hybridize;

(d) removing the hybridized repetitive sequences of the human DNA and the probe from the mixture; and

(e) recovering the remainder of the probe from the mixture in the form of a nucleic acid probe having a substantial quantity of the repetitive sequences absent therefrom.

2. A method as claimed in claim 1, in which the human DNA is comprised of amplified, microdissected fragments.

3. A method as claimed in claim 2, in which said fragments are labeled with biotin by nick translation.

4. A method as claimed in claim 1, in which in step (c) the reaction is hybridization of microdissected DNA fragments and the probe is biotin-labeled human repetitive sequences.

5. A method as claimed in claim 1, in which in step (d) the hybridized repetitive sequences are removed by incubating the product of step (c) with avidin and subtracting them with phenol .

6. The method claimed in claim 5, in which after phenol subtraction, the hybridized repetitive sequences remain in solution after the addition of acetate and alcohol .

7. The method claimed in claim 1, in which in step (d) the remainder of the probe is recovered by PCR using a primer.

8. A product produced by the process of claim 1.

9. The product produced by the process of claim 7.

10. A process of staining target chromosomal DNA with a nucleic acid probe to identify genomal rearrangements without at least a portion of the distraction of hybridization of repetitive sequences in both the target DNA and the probe, comprising:

(b) providing target chromosomal DNA containing repetitive sequences that hybridize with said repetitive sequences of the probe;

(c) also providing human DNA containing repetitive sequences that hybridize with the reptitive sequences of the probe;

(d) reacting a mixture of said human DNA and said probe so that the reptitive sequences of the human DNA and the probe hybridize;

(e) removing said hybridized repetitive sequences of human DNA and said probe from the mixture;

(f) recovering the remainder of the probe from the mixture in the form of an improved nucleic acid probe having a substantial quantity of the repetitive sequence removed therefrom, and

(g) applying said improved probe to said target chromosomal DNA, whereby said probe will bind to said target DNA but repetitive sequences of said target DNA will not have complementary repetitive sequences of said probe to which to bind and mitigate the effectiveness of the probe in identifying specific genomal rearrangements in said target chromosoma1 DNA.

11. The method claimed in claim 10, in which step (d) the reaction is hybridization of microdissected DNA fragments and the probe is biotin-labeled human repetitive sequences.

12. The product produced by the process of claim 10.

13. The product produced by the process of claim 11.