Production of chromosome region specific DNA sequences and transamination

• US 5,506,350 A
• Filed: 08/19/1993
• Issued: 04/09/1996
• Est. Priority Date: 09/20/1990
• Status: Expired due to Term

• Current Assignee: Clarissa F. Prorok, Kenneth A. Cruickshank, Lucy M. Stols, Michael L. Bittner
• Original Assignee: Clarissa F. Prorok, Kenneth A. Cruickshank, Lucy M. Stols, Michael L. Bittner
• Inventors: Cruickshank, Kenneth A., Prorok, Clarissa F., Stols, Lucy M., Bittner, Michael L.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): CAMPBELL, EGGERTON A
• Application Number:
  US08/109,167
• Time in Patent Office: 964 Days
• Field of Search: 536/55.3, 536/55.1, 536/23.1, 536/25.32, 435/6
• US Class Current: 536/55.3
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

Techniques for producing cloned DNA sequences are provided which sequences are complementary to DNA occurring in one selected region of one chromosome of a multi-chromosomal genome, such as the human genome. Such cloned DNA sequences can be labeled and formed into probes by conventional procedures, there are provided methods for making probe compositions which comprise mixed DNA segments derived from such a DNA sequence. An improved DNA sequence transamination procedure is provided utilizing trifluoroacetate chaotrope anions. With high concentrations of low complexity DNA, high levels of transamination are thereby achieved. These segments are covalently bound to fluorophore groups through linking groups that are transaminated preferably chaotropically into the segments.

Claims

1. In an improved process for transaminating a polynucleotide under aqueous liquid phase conditions, said polynucleotide containing at least one deoxycytidine nucleotide per molecule, in the presence of a bisulfite catalyst and a reactive compound comprising the formula:

##STR6## wherein;

X is a divalent radical selected from the class consisting of ##STR7## R is an alkylene radical containing 2 through 12 carbon atoms inclusive, and

R¹ and R² are each independently selected from the class consisting of hydrogen and lower alkyl, the improvement which comprises carrying out the transaminating in the presence of a dissolved trihaloacetate chaotrope anion while maintaining a temperature of 20°

to 60°

C. and a pH of 4.5 to 7.5.

2. The process of claim 1 wherein said trihaloacetate chaotrope anion is selected from the group consisting of trifluoroacetate and trichloroacetate.

3. The process of claim 1 wherein said trihaloacetate chaotrope anion is trifluoroacetate.

4. The process of claim 1 which is carried out by the steps of:

(a) contacting said polynucleotide sequence in an aqueous solution containing as solutes (1) alkali metal bisulfite, and (2) at least one water soluble trihaloacetate salt wherein the trihaloacetate anion is selected from the group consisting of trifluoroacetate, trichloroacetate, and mixtures thereof, with a water soluble difunctional linking compound as said reactive compound, said linking compound containing two substituent functional radicals per molecule, one of which is selected from the group consisting of primary and secondary amino radicals, and the other of which is selected from the group consisting of primary amino radicals, secondary amino radicals, carboxylic acid radicals, and carboxylate radicals, said functional radicals each being bonded to an organic radical that contains at least two and not more than 20 carbon atoms;

(b) maintaining said solution at a pH of 4.5 to 7.5 and at a temperature of 20°

to 60°

C. until transamination of said deoxycytidine nucleotide(s) by an amino group of said linking compound occurs to a predetermined extent;

(c) dialyzing the resulting transaminated solution against an alkali metal lower alkanoate to separate therefrom substantially all non-alkanoate salt anions;

(d) precipitating from the so dialyzed solution the resulting transaminated nucleotide sequence product; and

(e) separating said so precipitated product.

5. The process of claim 4 wherein said difunctional linking compound is ethylene diamine.

6. The process of claim 4 wherein said polynucleotide is selected from the group consisting of (a) at least one DNA sequence produced by the process of claim 1 and (b) a mixture of fragmented segments derived therefrom.

7. The process of claim 6 wherein

(a) said polynucleotide comprises said mixture of fragmented segments and said mixture is transaminated to an extent such that 12 to 70 mole percent of all deoxycytidine nucleotides present therein are so transaminated by said linking compound,

(b) said segments have average sizes of 150 to 600 base pairs,

(c) the concentration of said segments in said solution is at least 20 micrograms per milliliter, and

(d) said segments contain not less than about 1×

10¹⁰ complementary sequential copies per microgram of said segments.

8. The process of claim 4 wherein the resulting so transaminated nucleotide sequence product is reacted with a label group containing compound which incorporates a reactive radical that is reactive with a functional radical in said resulting so transaminated nucleotide sequence product that is derived from said linking compound.

9. In an improved process for transaminating a polynucleotide under aqueous liquid phase conditions, said polynucleotide containing at least one deoxycytidine nucleotide per molecule, in the presence of a bisulfite catalyst and a reactive compound comprising the formula:

##STR8## wherein;

X is a divalent radical selected from the class consisting of ##STR9## R is an alkylene radical containing 2 through 12 carbon atoms inclusive, and

R¹ and R² are each independently selected from the class consisting of hydrogen and lower alkyl, the improvement which comprises carrying out the transaminating in the presence of a trifluoroacetate anion while maintaining a temperature in the range of 20°

C. to 60°

C. and a pH in the range of 4.5 to 7.5.

10. The method of claim 9 wherein pH is in the range of 6.5 to 7.5.

11. In an improved process for transaminating a polynucleotide under aqueous liquid phase conditions, said polynucleotide containing at least one deoxycytidine nucleotide per molecule, in the presence of a bisulfite catalyst and a reactive compound comprising the formula:

##STR10## wherein;

X is a divalent radical selected from the class consisting of ##STR11## R is an alkylene radical containing 2 through 12 carbon atoms inclusive, and

R¹ and R² are each independently selected from the class consisting of hydrogen and lower alkyl, the improvement which comprises carrying out the transaminating in the presence of a trichloroacetate anion at a concentration of less than 1M while maintaining a temperature in the range of 20°

C. to 60°

C. and a pH in the range of 4.5 to 7.5.

12. The method of claim 11 wherein pH is in the range of 6.5 to 7.5.