Solid phase nucleic acid labeling by transamination

Details Solid phase nucleic acid labeling by transamination Solid phase nucleic acid labeling by transamination

1999-03-26

2001-02-27

Riley, Jezia (Department: 1655)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S006120, C536S022100, C536S023100, C536S025300, C536S025400

Reexamination Certificate

active

06194563

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to nucleic acid labeling chemistry.
BACKGROUND OF THE INVENTION
The labeling of nucleic acids with detectable probes has been a common molecular biology laboratory technique for decades. In recent years, the development and commercial success of nucleic acid microarrays (sometimes also called biochips) has often relied, in part, on the use of labeled nucleic acid probes.
Many nucleic acid labeling techniques require indirect labeling; in other words, the nucleic acids are labeled with one member of a specific binding pair (e.g., biotin), hybridized to a target nucleic acid, and visualized with the other member of the specific binding pair which is detectably labeled (e.g., horseradish peroxidase-labeled streptavidin). Direct labeling of nucleic acids has recently been achieved using bisulfite-catalyzed transamination (BCT) of cytidines within a nucleic acid (U.S. Pat. Nos. 5,506,350 and 5,491,224). However, these direct labeling procedures, performed in solution, are limited by the time (two or more days) required to achieve sufficient labeling of the nucleic acids.
Oligonucleotides are commonly made today using a solid phase methodology in which the oligos are prepared by successive iterative additions of nucleoside monomers while the growing oligonucleotide chain is attached to a solid phase. Oligonucleotides have been modified with single or multiple fluorescent dye molecules while still attached to the synthesis solid phase, e.g., as described in Haralambidis et al.,
Nuc. Acids Res.,
18:501-505, 1990. However, this methodology cannot be applied to complex genomic DNAs such as that extracted from solid tumor samples or blood samples.
SUMMARY OF THE INVENTION
The invention is based on the discovery that nucleic acids immobilized on a solid support can be efficiently and rapidly functionalized with reactive groups by a transamination reaction (e.g., a bisulfite-catalyzed transamination [BCT] reaction). Subsequent to the transamination, the functional groups (typically primary amino groups) can be derivatized with a fluorescent dye and released from the solid phase using a variety of chemical reactions. In general, these reactions should have one or more of the following characteristics: (1) not compromise the fluorescence of the dye, (2) not damage the hybridization properties of the DNA probe, and (3) give high yields of fluorescently labeled nucleic acid product.
A prominent aspect of the invention is the reversibility of the attachment chemistry. For example, efficient attachment of nucleic acids can be achieved using phosphoramidate linkages that are stable to the BCT and dye attachment chemistries, yet permit efficient release of the nucleic acid from the solid support under conditions of elevated pH for DNA stability. Since RNA can be hydrolyzed under elevated pH conditions, the releasing step in labeling RNA may require an alternative release chemistry such as thermal formamide treatment.
Accordingly, the invention features a method for linking a detectable label to a nucleic acid by (1) providing a nucleic acid bound (e.g., covalently bound) to a solid support (e.g., a bead or a chromium-plated glass slide), the nucleic acid including a cytidine base; (2) transaminating (e.g., by a bisulfite-catalyzed reaction) the cytidine base with a reactive group to form a covalent linkage between the cytidine base and the reactive group; and (3) linking a detectable label to the reactive group.
The reactive group can be of the formula:
where R is an alkylene radical containing 2 to 14 carbon atoms inclusive, X is
R
1
and R
2
are each independently selected from the group consisting of hydrogen and a lower alkyl. Further, the reactive group can be H
2
N(CH
2
CH
2
X)
n
CH
2
CH
2
NH
2
, where n=1-4, X is O, SO, SO
2
, Si, or NR
3
, where R
3
is a lower alkyl. An example of a reactive group is ethylenediamine.
In the method, the reactive group can be dissolved in a solvent which includes a trihaloacetate chaotrope anion (e.g., trifluoroacetate), the detectable label can be a fluorescent molecule, and the nucleic acid can be about 1 to 2 kilobases in length.
The method optionally includes releasing the nucleic acid from the solid support.
The nucleic acid can be bound to the solid support by reacting a terminal phosphate of a nucleic acid and an amino activated solid support with 1-ethyl-3-(dimethylaminopropyl)carbodiimide.
In another aspect, the invention includes a composition having (1) a solid support; (2) a nucleic acid bound to the solid support, the nucleic acid including a cytidine base; (3) a reactive group covalently linked to the cytidine base; and (4) a detectable label linked to the reactive group (e.g., a bead or a chromium-plated glass slide).
The reactive group includes the formula:
where R is an alkylene radical containing 2 to 14 carbon atoms inclusive, X is
R
1
and R
2
are each independently selected from the group consisting of hydrogen and a lower alkyl. Further, the reactive group can be H
2
N(CH
2
CH
2
X)
n
CH
2
CH
2
NH
2
, where n=1-4, X is O, SO, SO
2
, Si, or NR
3
, where R
3
is a lower alkyl. An example of a reactive group is ethylenediamine.
In addition, the detectable label can be a fluorescent molecule, the nucleic acid can be about 1 to 2 kilobases in length, and the nucleic acid can be covalently bound to the solid support. The nucleic acid can be bound to the solid support by reacting a terminal phosphate of a nucleic acid and an amino activated solid support with 1-ethyl-3-(dimethylaminopropyl)carbo-diimide.
In another aspect, the invention includes a method for linking a detectable label to a plurality of nucleic acids, each of the plurality having a unique sequence, by (1) providing the plurality of nucleic acids bound to a solid support, each of the plurality including a cytidine base; (2) transaminating each cytidine base with reactive groups to form a covalent linkage between each cytidine base and a reactive group; and (3) linking a detectable label to the reactive groups.
In yet another aspect, the invention includes a kit containing (1) a solid support (e.g., a bead), (2) a bisulfite, (3) a reactive group; and (4) a detectable label.
The solid phase BCT labeling procedure described herein can be performed in a matter of hours, a time frame unexpectedly shorter than that required for solution BCT labeling. The new labeling techniques are especially useful for automated or robotic labeling of large numbers of DNA samples. In addition, the performance of solid phase BCT labeling of DNA can be facilitated by kits that include a solid support, a detectable label, and BCT reagents required for labeling.
The development of biochips has reduced the time required for global analysis of a DNA sample by enabling simultaneous measurement of the expression of thousands of genes. However, the total analysis time is, in many cases, limited by the time required to directly label the sample DNA. Thus, by enabling highly efficient and rapid direct labeling of nucleic acids, the methods and kits of the invention further decrease the time required for global DNA analysis.
In addition, the compositions of the invention provide a novel means to analyze a DNA sample. For example, a fluorescently labeled DNA sample immobilized on a solid support can be hybridized to probe DNA containing a different fluorescent label. The presence of a sequence which hybridizes to the probe is then detected by a change in fluorescence color.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although suitable methods and materials for the practice or testing of the present invention are described below, other methods and materials similar or equivalent to those described herein, which are well known in the art, can also be used. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case o

Affiliated with

Also associated with

Rating

Solid phase nucleic acid labeling by transamination does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Solid phase nucleic acid labeling by transamination, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Solid phase nucleic acid labeling by transamination will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2566534