Method of designing primer and probe sets, primer and probe set designed by the method, kit comprising the sets, computer readable medium recorded thereon program to execute the method, and method of identifying target sequence using the sets

• US 20060204996A1
• Filed: 03/06/2006
• Published: 09/14/2006
• Est. Priority Date: 03/08/2005
• Status: Active Grant

• Current Assignee: Samsung Electronics Co. Ltd.
• Original Assignee: Samsung Electronics Co. Ltd.
• Inventors: Lee, Kyu-sang, Oh, Ji-young, Kwon, Tae-joon
• Granted Patent: US 7,657,382 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

A method of designing primer and probe sets for identification of target sequences by amplification and hybridization is provided. Also, primer and probe sets designed by the method, a kit comprising the sets, a computer readable medium recorded thereon a program to execute the method, and a method of identifying target sequences using the sets are provided. Accordingly, primer and probe sets capable of identifying rapidly and accurately a number of target sequences can be readily designed.

Claims

1. A method of designing primer and probe sets for identification of a target sequence by amplification and hybridization, comprising:

(a) preparing subsequence sets by cleaving a plurality of target sequences according to a predetermined criterion while sliding 1 bp by 1 bp so as to include all possible subsequences;

(b) selecting two subsequence sets, in which the number of subsequences having homology greater than a predetermined level is greatest, by comparing the subsequence sets;

(c) selecting subsequences which have homology greater than a predetermined level in the two subsequence sets as a common subsequence set and selecting the remaining subsequences as probes;

(d) repeatedly performing the operations (b) and (c) on the subsequence sets (excluding the two subsequence sets) and the common subsequence set until there are no subsequences having homology greater than a predetermined level between subsequence sets; and

(e) selecting subsequences of common subsequence sets which contain no subsequences having homology greater than a predetermined level as primers.

2. The method of claim 1, wherein the criterion in the operation (a) is at least one selected from the group consisting of a base length, a hybridization melting point (Tm), a GC content, self-alignment, a mutation position, a repeating sequence level, and a base composition at the 3′

end.

3. The method of claim 1, wherein the selecting of two subsequence sets in the operation (b) is carried out considering thermodynamic characteristics and position information.

4. Primer and probe sets designed using the method of claim 1.

5. A microarray in which the probe set of claim 4 is immobilized on a substrate and a kit for identification of a target sequence, comprising the primer set of claim 4.

6. The microarray of claim 5, wherein the substrate is coated with an active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde.

7. The microarray of claim 5, wherein the substrate is a silicon wafer, glass, quartz, metal, or plastic.

8. A computer readable medium recorded thereon a program to execute the method of claim 1.

9. A method of identifying a target sequence using the primer and probe sets of claim 4.

10. The method of identifying a target sequence, which comprises:

amplifying a target sequence using the primer set of claim 4;

hybridizing the amplified target sequence with the probe set of claim 4;

washing and removing a non-specific reaction; and

detecting a fluorescent signal due to hybrid formation.