JP2004523221A - Genotyping using partially complementary probes - Google Patents

Abstract

Methods are provided for detecting at least two different target sequences in a test sample. Such methods use probes that are not perfectly complementary to each other (eg, SNPs), but nevertheless hybridize to each other, to detect different target sequences. The target sequence can be amplified before detection by the probe. Single sequence variant forms are particularly suitable for detection by the methods provided herein.

Description

【００４８】
５’末端の２個の塩基を除去して、脱安定化のためのミスマッチＧと共に（配列番号７、Ａ）又はミスマッチなしで（配列番号１１、Ｅ）３’末端に付加塩基を加えて、あるいは５’末端でさらに２個の塩基を欠失させるが３、４又は５個の塩基を３’末端に加えて（それぞれ配列番号８（Ｂ）、９（Ｃ）又は１０（Ｄ））、突然変異体プローブへの修飾を行った。配列番号５の野生型プローブによる別個の反応において、配列番号６の代わりにこれらの突然変異体プローブの各々を使用して上述した実験手順を反復した。結果を下記の表２に示す。
【００４９】
【表２】

【００５０】
すべての突然変異体プローブは先の配列番号６突然変異体プローブよりも良好な成績を示し、ヘテロ接合及びホモ接合突然変異体ＤＮＡの両方を検出する能力を有していた。突然変異体プローブＤを除いて、突然変異体プローブはまた、野生型ＤＮＡを検出しなかったことから、良好な特異性を示した。野生型プローブは引き続き良好な成績を収め、野生型及びヘテロ接合ＤＮＡの両方を検出したが、ホモ接合突然変異体ＤＮＡは検出しなかった。最良のシグナルは、突然変異体プローブＣ（配列番号９）及びＤ（配列番号１０）を用いて達成された。従って、野生型又は突然変異体ライデン第Ｖ因子ＤＮＡを検出するためのプローブの最良の組合せは、５’末端に２塩基の欠失を有する野生型プローブ（配列番号５）と、やはり５’末端に２塩基の欠失を有するが３’末端に４個の付加塩基を含む突然変異体プローブＣ（配列番号９）を使用することであった。これは、野生型と突然変異体ライデン第Ｖ因子配列間の塩基対ミスマッチを中央から５’末端近くに移動させて、塩基対ミスマッチを有効に相殺する。
【００５１】
それ故、概して、オフセットプローブはそれらの適切な増幅標的配列に特異的にハイブリダイズすることができた。
【００５２】
実施例５
突然変異体対野生型プロトロンビンの検出
Ａ．センス鎖プローブの使用：プロトロンビン遺伝子上の野生型対突然変異体多型を検出するための初期実験を、別々の反応において同様のセンス鎖プローブ、一方は突然変異体遺伝子（配列番号１６）についてのプローブ、他方は野生型遺伝子（配列番号２０）についてのプローブを使用して実施した。野生型対突然変異体遺伝子座における一塩基対の相違に加えて、野生型プローブは同時に５’末端に２個の付加塩基対を含んだ。
【００５３】
精製ヘテロ接合及びホモ接合突然変異体ライデン第Ｖ因子／プロトロンビンＤＮＡ試料のデュープリケートをＭｏｌｅｃｕｌａｒ Ｇｒａｄｅ Ｗａｔｅｒ（５’−３’，Ｉｎｃ．，Ｂｏｕｌｄｅｒ，ＣＯ）中で１：１００、１：１０００及び１：１０，０００に希釈し、アダマンタンで標識した配列番号１４及び配列番号１５プライマーを用いてＰＣＲ増幅して、別々の反応においてカルバゾール標識した配列番号１６突然変異体プローブ又はカルバゾール標識した配列番号２０野生型プローブを用いて検出した。ＰＣＲは、５０ｍＭ Ｂｉｃｉｎｅ（Ｎ，Ｎ−ビス［２−ヒドロキシエチル］グリシン）、ｐＨ８．１、１５０ｍＭカリウム、８％ｗ／ｖグリセロール、０．００１％ウシ血清アルブミン（ＢＳＡ）、０．１ｍＭ ＥＤＴＡ及び０．０２％アジ化ナトリウムの最終濃度を含む緩衝液中で実施した。各々１５０μＭの最終濃度で存在するｄＮＴＰ（ｄＡＴＰ、ｄＧＴＰ、ｄＴＴＰ及びｄＣＴＰ）と共に、組換えＴｈｅｒｍｕｓ ｔｈｅｒｍｏｐｈｉｌｕｓポリメラーゼを５単位／反応の濃度で使用した。プライマーは各々２５０ｎＭの濃度で使用し、プローブは各々５ｎＭの濃度で存在した。２０μｌの試料容量に関して、３．２５ｍＭ塩化マンガンの最終濃度を０．２ｍｌの総反応容量中で使用した。２０ｎｇ／μｌのポリ−Ａの対も陰性対照として試験した。
【００５４】
反応混合物をＬＣｘ（登録商標） Ｔｈｅｒｍａｌ Ｃｙｃｌｅｒにおいて増幅した。反応混合物を最初に９５℃で４分間、次に５６℃で３０秒間、さらに９７℃で２分間インキュベートし、その後９５℃で４０秒間、次いで５８℃で６０秒間の４０サイクルのＰＣＲ増幅に供した。反応混合物を熱サイクルに供した後、混合物を７２℃に５分間、次いで９７℃に５分間保持し、２分間温度を１５℃に下げてプローブオリゴハイブリダイゼーションを実施した。その後反応産物を分析し、検出するときまで、試料を４℃に保持した。
【００５５】
反応産物をＡｂｂｏｔｔ ＬＣｘ（登録商標）システム（Ａｂｂｏｔｔ Ｌａｂｏｒａｔｏｒｉｅｓ，Ａｂｂｏｔｔ Ｐａｒｋ，ＩＬより入手可能）で検出した。抗カルバゾール被覆微粒子の懸濁液及び抗アダマンタン抗体／アルカリホスファターゼコンジュゲート（すべてＡｂｂｏｔｔ Ｌａｂｏｒａｔｏｒｉｅｓ，Ａｂｂｏｔｔ Ｐａｒｋ，ＩＬより入手可能）をＬＣｘ（登録商標）と共に使用して反応産物を捕獲し、検出した。使用した酵素基質は４−メチル−ウンベリフェリルホスフェート（ＭＵＰ）であり、基質から生成物への変換速度を測定し、計数／秒／秒（ｃ／ｓ／ｓ）として報告した。
【００５６】
下記の表３からわかるように、突然変異体プローブはヘテロ接合とホモ接合突然変異体試料の両方を検出し、野生型プローブは、ヘテロ接合プローブだけと反応し、ホモ接合突然変異体とは反応しないはずであるにもかかわらず、やはり両方の試料を検出した。それ故、どちらもセンス鎖からのプローブであるこのプローブの組合せは、プロトロンビン遺伝子の突然変異体多型から野生型を区別することができなかった。
【００５７】
【表３】

【００５８】
Ｂ．センス対アンチセンスプローブの使用：センス鎖からのプローブはプロトロンビン遺伝子の突然変異体対立遺伝子から野生型を区別することができなかったので、野生型プローブをアンチセンス鎖に移して、これが２つの対立遺伝子の識別を容易にするかどうかを調べた。
【００５９】
この実験は、配列番号２０プローブの代わりにカルバゾール標識した配列番号１７野生型プローブを使用したこと及び試料のデュープリケートを１：１００希釈だけで試験したことを除いて、上記の実施例５．Ａ．で述べたように実施した。熱サイクリング条件も次のように変更した：反応混合物を最初に９７℃で２分間インキュベートし、次いで９５℃で４０秒間、５８℃で６０秒間の４０サイクルのＰＣＲ増幅に供した。反応混合物を熱サイクルに供した後、混合物を５分間９７℃に保持し、２分間温度を１５℃に下げてプローブオリゴハイブリダイゼーションを実施した。その後反応産物を分析し、検出するときまで、試料を１５℃に保持した。
【００６０】
【表４】

【００６１】
上記の表４の結果は、野生型プローブをアンチセンス鎖に移すことによって野生型と生じた突然変異体対立遺伝子間の識別が可能になり、野生型プローブが、ホモ接合突然変異体試料に関してよりも、１個の野生型対立遺伝子を含むヘテロ接合試料に関してはるかに高い検出シグナルを示すことを明らかにしている。突然変異体プローブはまた、ヘテロ接合とホモ接合突然変異体試料の両方を検出する能力も保持している。さらに、これらの条件下で突然変異体プローブは、実施例５．Ａ．の実験で認められたものに比べて、１個の突然変異体対立遺伝子だけを含むヘテロ接合試料よりも２個の突然変異体対立遺伝子を含むホモ接合突然変異体試料に関してわずかに高いシグナルを生じる。
【００６２】
Ｃ．オフセットプローブの使用：上記の実施例５．Ｂ．において、センス鎖上の一方のプローブとアンチセンス鎖上の他方のプローブを使用して野生型と突然変異体プロトロンビンの識別が達成されたが、遺伝子型を決定するために試料を１回しかし検査しなくてもよいように、１つの反応混合物中で両方のプローブを組み合わせられることは好都合である。この実験も別々の反応においてセンス（突然変異体）／アンチセンス（野生型）プローブを使用したので、試料の遺伝子型を決定するために各々の試料について２つの試験を実施する必要があった。しかし、センス／アンチセンスプローブは一塩基対ミスマッチを除いて互いの正確な相補物であるので、それらはまた、それらの特異的標的増幅ＤＮＡとではなくお互いとハイブリダイズしうる。これを防ぐのを助けるために熱サイクリング条件を修正し、加えて、ライデン第Ｖ因子プローブに関して使用した、塩基対ミスマッチ付近でプローブを相互からオフセットするという戦略を利用した。
【００６３】
プロトンビン遺伝子の突然変異体対立遺伝子を検出するためのプローブ（配列番号１６）は、上記の実施例５．Ａ．及びＢ．で使用したものと同じであり、中央（１３塩基対のうちの塩基対６）に塩基対ミスマッチを含んだ。３個の野生型プローブをこの突然変異体プローブと共に試験した：野生型プローブＦ（配列番号１９）は一塩基対ミスマッチを除いて突然変異体プローブと相補的であった；野生型プローブＧ（配列番号１７）は３’末端に２個の付加塩基対を含んだ；および野生型プローブＨ（配列番号１８）は３’末端に２個の付加塩基対を含み、５’末端から２個の塩基を欠失しており、各々のプローブの３’末端に２塩基突出を創造していた。
【００６４】
精製野生型、ヘテロ接合及びホモ接合突然変異体ライデン第Ｖ因子／プロトロンビンＤＮＡ試料の各々のデュープリケートをＰＣＲ増幅し、別々の反応において配列番号１４と配列番号１５のプライマー、及び配列番号１６の突然変異体プローブを上述した３個の野生型プローブの各々と共に使用した（すなわち、同じ反応混合物中で突然変異体プローブと野生型プの各々１個を使用した）ことを除いて実施例４におけるように検出した。使用した反応条件は、プライマーとプローブの濃度が次のとおりであったことを除いて、上記の実施例４で述べたとおりであった：配列番号１４のプライマー（カルバゾールで標識）を２００ｎＭの濃度で使用し、配列番号１５のプライマーを１５０ｎＭの濃度で使用し、配列番号１６の突然変異体プローブを７０ｎＭの濃度で使用し、配列番号１７（Ｇ）、１８（Ｈ）又は１９（Ｆ）の野生型プローブのいずれかが４５ｎＭの濃度で各反応混合物中に存在した。この実験からの結果を下記の表５に示す。
【００６５】
【表５】

【００６６】
すべての野生型／突然変異体プローブ対が適切な野生型又は突然変異体対立遺伝子の特異的検出を示し、野生型プローブは、ホモ接合野生型とヘテロ接合プロトロンビン試料を検出したが、ホモ接合突然変異体プロトンビンＤＮＡを陽性として検出しなかった。突然変異体プローブは、ホモ接合突然変異体とヘテロ接合プロトロンビン試料を検出したが、ホモ接合野生型プロトンビン試料を陽性として検出しなかった。予想されたように、ヘテロ接合試料は１個の野生型と１個の突然変異体対立遺伝子を含むので、すべてのプローブが用量依存的にヘテロ接合試料を検出した。従って、すべてのプローブが高い特異性を示した。
【００６７】
突然変異体プローブに完全に相補的である（一塩基対ミスマッチを除いて）野生型プローブ（Ｆ）は機能したが、ホモ接合野生型試料に関して最も低いシグナルを生じた。野生型対立遺伝子を検出する際に最良のシグナルを示した野生型プローブは突然変異体プローブＨ（配列番号１８）であり、両方の末端に２塩基対突出を生じるプローブの組合せであった。このプローブの組合せはまた、突然変異体と野生型陽性シグナル間の最良のバランス（等価性）を示した。
【００６８】
特定実施形態を参照して本発明を詳細に説明したが、本発明の精神と範囲から逸脱することなくそのような実施形態に様々な変更と修正を加えることは当業者には明白である。[0001]
(Technical field)
The present invention relates to detecting nucleic acid sequences, and in particular, to detecting different nucleic acid sequences with probes that are complementary to such different nucleic acid sequences and simultaneously complementary to each other.
[0002]
(Background of the Invention)
Tests designed to determine the genomic sequence of various organisms, as well as those designed to compare genomic sequences, have provided information about polymorphisms in various genomes, from the human genome to the HIV genome. A great variety of polymorphisms in the genome of such organisms have been reported. Various types of genetic polymorphisms include single nucleotide substitutions, insertions or deletions, various numbers of tandem repeats, deletions of all or most of the gene, and chromosomal rearrangements. Generally, polymorphisms involving a single nucleotide are called single nucleotide polymorphisms or "SNPs" (snips). If the genome contains a particular SNP, the sequence from the region containing the SNP may be present in one of two or more forms. If one of the forms can be identified as occurring in the majority of the population, or can cooperate with a protein product that retains full function, it is referred to as a "wild-type" sequence. Other forms of SNPs are called "mutants."
[0003]
Some mutations in a gene cause various disease aspects. For example, venous thrombosis is a fairly common disease that affects approximately 1 in 1000 people annually. In some cases, thrombosis was found to be SNPs in genes encoding proteins involved in a cascade of events responsible for blood coagulation. For example, a mutation in the gene encoding factor V, the so-called "Factor V Leiden" mutation, has been reported to be a causative agent of thrombosis. In addition, mutations in the 3 'untranslated region of the prothrombin gene have been reported to cause thrombosis. Individuals with either or both of the above genetic mutations have been reported to be at even greater risk of thrombosis, for example, when undergoing surgery, becoming pregnant, or taking oral contraceptives.
[0004]
In some cases, the mutant and wild-type sequences may be contained within a single genome. Such a situation can occur, for example, if the sequence is derived from an organism containing more than one set of chromosomes (sometimes referred to as "ploidy"). In addition, an organism containing one chromosome may simultaneously contain two or more mutated forms of the gene if that chromosome contains gene amplification. While the genome may contain duplicate copies of either the wild-type or mutant version of the sequence in question (sometimes referred to as "homozygous" for a particular sequence), the genome may be wild-type or mutant. There is an additional possibility of including one copy of each of the sequences (sometimes referred to as "heterozygosity" for a particular sequence). When trying to detect these sequences in a test sample, the presence of closely related sequences presents several challenges.
[0005]
Heretofore, detection of related sequences in test samples has been performed using RFLP and gel electrophoresis. Separate, separate amplification reactions to determine the presence of mutant and wild-type sequences in a sample have also been performed to detect the presence of multiple related sequences in a test sample. For example, genetic tests for Factor V Leiden and prothrombin mutations have been reported in the literature. Many of the gene-based assays reported to date have been performed utilizing amplification reactions such as the polymerase chain reaction (PCR) or the ligase chain reaction (LCR). After amplifying a region suspected of containing a specific mutation, the amplification product is often detected using gel electrophoresis. The amplification-based assay for mutations described above separately performs one reaction designed to detect the wild-type sequence and another reaction designed to detect the mutant sequence. Therefore, these assays are cumbersome and expensive to perform. Therefore, it would be useful to provide assays and assay reagents that can perform assays for two related sequences in one reaction vessel. Such reagents and methods would therefore avoid the use of binary separate reactions and the use of gel electrophoresis to detect relevant sequences in a test sample.
[0006]
(Brief description of the invention)
The present invention provides a method for detecting at least one of two related target nucleic acid sequences in a test sample. The method can be used with nucleic acid amplification techniques that can be conveniently performed in one reaction vessel. Generally, the method includes contacting a test sample with first and second probes. The probes are (i) completely complementary to each other except for at least one mismatch, and (ii) the first probe is more complementary to the sense strand of the target sequence than to the second probe. , (Iii) the second probe is more complementary to the antisense strand of the second target sequence than to the first probe, and (iv) the probes are designed to hybridize to each other at ambient temperature. A target sequence / probe hybrid is formed by hybridizing at least the first or second probe to the first or second target sequence. The hybrid so formed is detected as an indicator of the presence of one of the relevant target sequences in the test sample. Also provided are reagents for performing the above methods.
[0007]
(Detailed description of the invention)
The present invention provides a method for detecting two different target sequences in a test sample using a pair of probes that hybridize to each other but are not completely complementary to each other. Generally, the method involves amplifying the target sequence, if necessary, using any of the well-known amplification techniques, and detecting the amplified target sequence, if present, with a probe. Advantageously, different target sequences can often be amplified using a common amplification reagent. Under certain circumstances, such as when the target sequence is present at a sufficient concentration, the target sequence may not require amplification before detection with the probe. Probes are generally designed to be not completely complementary to their respective target sequences, but sufficiently complementary to hybridize to one another. Typically, the probes will have at least one mismatched base pair, preferably located between the 5 'and 3' terminal nucleotides of each probe. The use of probes that are more complementary to each target sequence than to each other allows the use of different but related target sequences in one test sample without having to introduce separate probes into the products of separate amplification reactions. It was found to allow detection. The invention is particularly suitable for detecting the presence of a mutant form of a nucleic acid sequence in a test sample.
[0008]
As used herein, "target sequence" means a nucleic acid sequence that is detected, amplified, amplified and detected, or is complementary to one of the amplification primers or probes. In addition, although the term target sequence is often used herein to refer to single-stranded nucleic acids, one of skill in the art will recognize that the target sequence may be double-stranded. As noted above, target sequences that can be detected using the methods provided herein are relevant. Such target sequences are related as long as one target sequence is usually a mutation of the other target sequence. In particular, target sequences that can be detected typically have the same sequence except that one of the target sequences contains a polymorphism that makes the sequence composition different from the other target sequence, for example. Thus, for example, a first target sequence and a second target sequence can be the same, except that the second target sequence contains one or more nucleotide substitutions, deletions, and / or insertions. The target sequence can be derived from any organism, including the nucleic acid genome.
[0009]
As used herein, the term "test sample" means anything suspected of containing the target sequence. Test samples include, for example, blood, bronchoalveolar lavage, saliva, throat swab, aqueous humor, cerebrospinal fluid, sweat, sputum, urine, milk, ascites, mucus, synovial fluid, intraperitoneal fluid, amniotic fluid, heart tissue Or any biological source such as fermented broth, cell cultures, chemical reaction mixtures and the like. The test sample can be used (i) directly from the source or (ii) after pretreatment to modify the properties of the sample. That is, the test sample may, for example, prepare plasma from blood, destroy cells, prepare liquid from solid material, dilute viscous liquid, filter liquid, distill liquid, concentrate liquid, interfere It can be pre-treated prior to use by inactivating components, adding reagents, purifying nucleic acids, and the like.
[0010]
“Primer sequence (s)”, “primer sequence (s)” or “primer (s)” are reagents that initiate the synthesis of multiple copies of a target sequence, most typically short nucleic acids or “oligos”. Nucleotide ". Oligonucleotides can vary widely in size, but typically range in length from 10 to 1000 nucleotides, more typically in the range from 15 to 100 nucleotides. Amplification reactions that use a primer sequence to synthesize multiple copies of a target sequence (or amplify the target sequence) are now well known in the art. A variant thereof, such as the LCR described in EP 320 308 and the gap LCR described in US Pat. No. 5,792,607 (hereby incorporated by reference), NASBA or US Pat. A similar reaction, such as TMA described in US Pat. No. 5,399,491, which is incorporated herein by reference, preferably PCR, and US Pat. Nos. 4,683,195 and 4,683,202. No. 5,310,652 (incorporated herein by reference in its entirety) are examples of amplification reactions that can be used in accordance with the present invention.
[0011]
As used herein, the phrase "amplification reaction reagent" refers to a reagent that is well known for use in a nucleic acid amplification reaction and may be single or multiple reagent (s), reagent (s), enzyme (s) or separately or Enzymes that individually have reverse transcriptase, polymerase, and / or ligase activity; enzyme cofactors such as magnesium or manganese; salts; nicotinamide adenine dinucleotide (NAD); and, for example, deoxyadenosine triphosphate; Including but not limited to deoxynucleoside triphosphates (dNTPs) such as deoxyguanosine triphosphate, deoxycytidine triphosphate and thymidine triphosphate. The exact amplification reagent to use is largely a matter of choice for one skilled in the art based on the particular amplification reaction used.
[0012]
Probe sequences are similar to primer sequences, as long as they hybridize to the target sequence, facilitating detection of the target sequence and are typically oligonucleotides. Probes are generally nucleic acids or uncharged alkyl analogs such as, for example, methylphosphonates and phosphotriesters, peptide nucleic acids (as disclosed in WO 93/25706), and morpholine analogs (US Pat. 5,142,047, 5,235,033, 5,166,315, 5,217,866 and 5,185,444). And nucleic acid analogs such as In addition, the term probe is used to refer to a portion of a nucleic acid sequence that is predetermined or designed to hybridize to its target sequence, as one skilled in the art can add other nucleotides to the probe for other purposes. It is intended to mean a contiguous group of nucleotides. For example, an extension or tail of such a probe is generally added for detection or purification of the probe.
[0013]
The probes are provided in pairs, each probe of a particular pair hybridizing to the other members of the pair. The probes, however, are not completely complementary to each other, meaning that there is at least one mismatched base pair on each probe. An "incorrect base pair" (sometimes simply referred to as a "mismatch") is one in which one or more nucleotides on one probe is typically one or more corresponding nucleotides on the other probe. Means no hydrogen bond in a typical Watson-Crick fashion. Generally, probes are more complementary to their respective target sequences than are they complementary to each other. Preferably, however, the probes are designed to be completely complementary to different target sequences, such that each nucleotide on an individual probe has a probe on its target sequence that completely matches the probe to its target. Find the corresponding complementary nucleotide to make it complementary.
[0014]
There are many possible configurations for the probe, and the exact configuration of the probe depends primarily on the particular target sequence chosen. In particular, mismatches between probes that make the probes partially non-complementary can occur at any site and can take any range depending on the length of the probe. However, the probes hybridize to each other at ambient temperature (eg, 15 ° C. to 30 ° C.) in the absence of their respective target sequences, but are configured to bind preferentially to their respective target sequences, if present. Should have been. Probes can be designed to hybridize to each other at ambient temperature using well-known hybridization principles or annealing kinetics, but preferentially bind to the target sequence when present. Hybridization depends fairly predictably on several parameters, including temperature, ionic strength, sequence length, complementarity, and the G: C content of the sequence. For example, lowering the environmental temperature of a complementary nucleic acid sequence will promote annealing. For a given set of sequences, the melting point or "Tm" can be assessed by several known methods. Ionic strength or "salt" concentration also affects the melting point because small cations tend to stabilize the formation of replicas by nullifying the negative charge on the phosphodiester backbone. Those skilled in the art will readily appreciate that typical salt concentrations depend on the nature and valency of the cation. Similarly, if the G: C content is high and the sequence is long, the A: T pair will contain only two hydrogen bonds, the G: C pair will contain three hydrogen bonds, and longer sequences will retain the sequence. It is known to stabilize the formation of replicas because they have more hydrogen bonds than possible. Therefore, the higher the G: C content and the longer the long sequences, the higher the Tm, affecting the hybridization conditions.
[0015]
Once the probes are selected for a given set of target sequences, the G: C content and length are known and can be a basis for accurately determining what hybridization conditions to include. The next most obvious and diverse parameter is temperature, since ionic strength is typically optimized for enzyme activity. Generally, the Tm of the hybridizing probe is lower than the Tm of the hybrid product formed between the probe and the respective target sequence. Obtaining a suitable probe configuration is therefore well within the state of the art for those skilled in the art. Further, determining whether a given probe configuration meets the above criteria can be performed empirically. For example, the hybridization properties for a particular set of probes and their respective target sequences can be determined by varying the environmental temperature of the probe and the environmental temperature of the probe and target. Generally speaking, the probes are selected to be more complementary to their respective targets, so that complementary probes will have a Tm that is essentially lower than the Tm between the probe and the respective target sequence.
[0016]
The exact size of the probe will depend primarily on the target sequence and the number of nucleotides involved in the mutation between the two sequences. Preferably, however, the probes are 10 to 1000 nucleotides in length, more preferably 10 to 100 nucleotides in length, and most preferably 10 to 50 nucleotides in length. The probes hybridize to each other except for the region containing the mutation between the two target sequences, each of which is specific for the probe. The region (s) of the non-complementary probe may be of any length, so long as the probe simultaneously has a region that is sufficiently complementary to hybridize at ambient temperature. Non-complementary regions of the probe can also be located at any site within the probe, as long as the probes hybridize to each other at ambient temperature. Typically, the probe will have, for example, a base substitution, deletion, or insertion involving, for example, 10 or less nucleotides, more typically less than 5 nucleotides, preferably less than 3 nucleotides, and most preferably 1 nucleotide polymorphism. Used to hybridize to target sequences containing "small mutations" or "small polymorphisms".
[0017]
Also, the region (s) of the non-complementary probe can be located in any region of the probe, except that one or both probes contain the overhangs detailed below, and the 5 'and 3''It is preferred to have these regions inside the terminal nucleotide. The terminal nucleotide is the last nucleotide at each end of the probe designed to hybridize to the target sequence. More preferably, the non-complementary region (s) of the probe is located between the terminal nucleotide and the central nucleotide (s) of the probe. As used herein, the term "central nucleotide" is intended to mean the nucleotide (s) located at the center of an individual probe. For example, in the case of a five nucleotide probe, the central nucleotide is the third nucleotide inside from the 5 'terminal nucleotide. In the case of a six nucleotide probe, the central nucleotide is the third and fourth nucleotides inward from the 5 'terminal nucleotide. Therefore, the region (s) of the non-complementary probe is preferably located away from the center (eg, near the 5 'end of one probe). It is particularly preferred that the mismatch be located away from the center when the mismatch is the result of a mutation between the target sequences and is the only mismatch between the probes.
[0018]
In view of the above, the configuration of several probe pairs is possible when the probes hybridize to each other. Preferably, the probes are not co-extensive unless the 3 'end of the probe is completely aligned with the 5' end of the complementary probe when the probes hybridize to each other. In other words, one probe is longer at one end than the other, which is commonly referred to in the art as an "overhang." Thus, an overhang at the 3 'end of a probe means that the probe extends beyond the 5' end of its complementary probe by one or more nucleotides. Preferably, one of the probes contains an overhang at the 3 'end. Alternatively, it is preferred to have a 3 'overhang on both probes.
[0019]
Although the probes are generally designed to be completely complementary to their respective target sequences, the probes are sufficient for the number of mismatches between the probes to meet the above criteria (i.e., at probe ambient temperatures to each other). As long as they hybridize, but preferentially bind to their respective targets). Probes may also have mismatches with the respective target sequences when they are functionalized, for example, with a nucleic acid tail that is not complementary to the target sequence. As mentioned above, the tail is typically used, for example, to detect hybrids formed between the probe and the respective target sequence.
[0020]
As mentioned above, the probes hybridize to different but related target sequences. In particular, the first probe hybridizes to the first target sequence and the second probe is complementary to the first target except for the presence of a mutation between the first and second target sequences, the first target sequence Hybridizes to the mutant form of Accordingly, probes are designed to hybridize to the opposite strand of the relevant target sequence. Thus, for example, if the target sequence is double-stranded, the first probe will be complementary to the sense strand of the first target sequence and the second probe will be complementary to the antisense strand of the second target sequence. Furthermore, despite the fact that the target sequence or its variant form may not be present in the test sample, the probe is pre-designed to hybridize to both forms of the target sequence. A variant form of the target sequence can include, for example, the same sequence as the first target sequence, including one or more nucleotide substitutions, deletions, and / or insertions. Thus, for example, if the target sequences hybridize to each other when they are the same except for a single nucleotide polymorphism, such as a single base pair substitution, the pair of probes may contain a single nucleotide mismatch.
[0021]
The probes provided herein can be used to detect various target sequences and mutations in such target sequences. For example, each target sequence to which the probe is complementary can be from a single-stranded RNA viral genome and a mutant or mutant strain of that particular viral genome. In particular, the first probe is complementary to a region of the single-stranded genome as normally occurs, and the second probe is complementary to the opposite strand of the same genome containing a mutation such as a single base pair substitution. sell. In this particular case, it will be apparent that the RNA is first reverse transcribed to DNA and the second probe hybridizing strand is present as a result of amplification of the reverse transcribed cDNA strand from the RNA genome. Further, according to this embodiment, only one version of the virus may be present in the test sample. Thus, only one probe will hybridize to its target.
[0022]
Alternatively, the original target sequence can be derived from the human genome. In this case, the first probe is complementary to the sense strand of the gene ("wild-type sequence") and the second probe is complementary to the antisense strand of the same gene ("mutant sequence") containing two single base substitutions. Can be complementary. Depending on the genotype of the individual sample, only the first probe will hybridize to its target if the genotype is homozygous for the wild-type sequence, and both the first and second probes if the genome is heterozygous. Will hybridize to their targets, and if the genome is a homozygous mutant, only the second probe will hybridize.
[0023]
Primers and / or probe sequences can be labeled using any of the well-known labels or labeling chemistry to detect hybrids formed between target sequences and probes, and can be selected primarily by those skilled in the art. Techniques for labeling sequences are well known and include, for example, those described in U.S. Patent Nos. 4,762,779 and 4,989,882, each of which is incorporated herein by reference. . Typically, the primer and probe sequences are labeled such that the hybrid (s) formed between the target sequence and the complementary probe can be identified. In other words, the complementary probe is labeled such that the first probe / target sequence hybrid can be distinguished from the second probe / target sequence hybrid. Various configurations for performing the above identifications are well known and can also be selected by those skilled in the art based on the selection of the detection device.
[0024]
As used herein, the term “label” refers to a molecule or moiety that has a detectable property or characteristic. The label can be directly detectable, eg, as in a radioisotope, fluorophore, chemiluminophore, enzyme, colloid particle, fluorescent microparticle, etc., or indirectly, eg, as in a specific binding member. Can be detected. It will be apparent that directly detectable labels may require additional components to allow detection of the label, such as, for example, substrates, triggering reagents, light, and the like. When using an indirectly detectable label, it is typically used in combination with a “conjugate”. The conjugate is a specific binding member typically attached or bound directly to a detectable label. Coupling chemistry for synthesizing conjugates is well known in the art, for example, without losing the specific binding properties of the specific binding member or the detectable properties of the label, and using some chemical and / or physical means. May be included. As used herein, a "specific binding member" is a member of a binding pair, i.e., two different molecules in which one molecule is specifically bound to the other molecule, for example, through chemical or physical means. Means a member. In addition to antigen-antibody specific binding pairs, other specific binding pairs include avidin and biotin, hapten and hapten specific antibodies, complementary nucleic acid or nucleic acid analog sequences, etc. It is not intended to be limited.
[0025]
Detection platforms that can be used to detect hybrids formed between complementary probes and their respective target sequences include any of the homogeneous or heterogeneous techniques well known in the art. An example of a homogeneous detection platform involves the use of a FRET label attached to a probe that emits a signal in the presence of the target sequence. The so-called TaqMan assay, described in US Pat. No. 5,210,015, incorporated herein by reference, is an example of a technique that can be used to detect nucleic acid sequences homogeneously. Briefly, for example, in a TaqMan fashion, one probe is combined with a first fluorophore that emits a signal at a first wavelength, and a suitable quenching unit that can quench the signal from the first fluorophore. Can be labeled with The second probe can be labeled with a second fluorophore that emits a signal at a second wavelength and a suitable quenching unit that can quench the signal from the second fluorophore. According to the principles of the TaqMan-type assay, the probe is cleaved in the presence of the respective target during an amplification reaction with an enzyme having a 5 'to 3' exonuclease activity. As a result, two different signals or no signal can be detected depending on the presence of the target.
[0026]
Heterogeneous formats typically use a capture reagent to separate the amplified sequence from other materials used to amplify and / or detect the target sequence. The capture reagent is typically a support material coated with one or more specific binding members specific for the same or different binding members. As used herein, "support" refers to a substance that is insoluble or can be made insoluble by a subsequent reaction. The carrier material may therefore be a latex, plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface or test tube surface, microtiter plate, sheet, bead, microparticle, chip, and other materials known to those skilled in the art. Other shapes are possible. Exemplary capture reagents include arrays comprising oligonucleotides or polynucleotides immobilized on a carrier material, generally in a spatially defined manner.
[0027]
For example, a heterogeneous format that can be used to detect a probe hybridized to a respective target sequence can include first and second probes labeled with first and second binding members. Primers used to amplify each target sequence can be labeled with a third binding member. If the target sequence is present, once the probe has hybridized to the target sequence to form the target sequence / probe hybrid, the hybrid can be immobilized on the carrier using the third binding member bound to the primer sequence. If desired, the support can be washed to remove excess reagent, and if a hybrid is present, the hybrid can be detected using a conjugate with a different directly detectable label. The signal bound to the solid phase, if present, can be detected to indicate the presence of the target sequence in the test sample.
[0028]
According to a preferred embodiment, "oligonucleotide hybridization PCR" (optionally) as described in US patent application Ser. No. 08 / 514,704, filed Aug. 14, 1995, which is incorporated herein by reference. Here, it is referred to as “OH PCR”. Briefly, the reagents used in the preferred method include primers, complementary probes, and amplification reagents for performing the amplification reaction. Primers are used to initiate extension of a copy of a target sequence (or its complement) and are labeled with a capture label or a detection label. The probe sequence is used to hybridize to a sequence generated by the primer, and typically to a sequence that does not include the primer. Like the primer, the probe is labeled with either a capture label or a detection label, except when the primer is labeled with a capture label, the probe is labeled with a detection label, and vice versa. A detection label has the same definition as a "label" defined above, and a "capture label" is typically used to separate extension products and probes bound to such products from other amplification reactions. . Specific binding members (as defined above) are very suitable for this purpose. Also, the probes used by this method are preferably blocked at their 3 'end so that they do not extend under hybridization conditions. Methods for preventing probe extension are well known and are a matter of choice for those skilled in the art. Typically, the addition of a phosphate group at the 3 'end of the probe is sufficient to block extension of the probe.
[0029]
According to the preferred embodiment described above, the probe is initially part of the reaction mixture. In addition, when the reaction mixture is placed under amplification conditions, a copy of the target sequence or its complement is produced at a temperature above the Tm of the probe, so that the primer, probe and probe have a lower melting point than the primer sequence. It is preferable to select amplification conditions. After such copies have been synthesized, they are denatured and the mixture is cooled to form a hybrid between the probe and the target or its complement. The rate of temperature decrease from the denaturation temperature to the temperature at which the probe binds to the single-stranded copy is preferably very rapid (eg, 8 to 15 minutes), especially enzymes with polymerase activity are active for primer extension. Temperature range. Such rapid cooling promotes copy sequence / probe hybridization rather than primer / copy sequence hybridization and extension.
[0030]
As indicated above, various target sequences and mutated forms of target sequences can be detected in one reaction vessel. For example, the portion of the gene encoding Factor V where the Factor V Leiden mutation occurs, and the Factor V Leiden mutation could be used as the target sequence and a variant form of the target sequence, respectively. Alternatively, the 3 'untranslated region of the prothrombin gene and the same region containing the mutation likely to contain the thrombosis-causing mutation could be used as the target sequence and a mutated form of the target sequence. Based on the sequence of these genes and known mutations, for example, a probe that is completely complementary to the sense strand of the wild-type sequence and the antisense strand of the mutant or mutant sequence, with overhangs as described above or It could be synthesized without overhang. Given the nature of the above mutations (ie, single base substitutions), such probes have at least one mismatch where the mutation occurs, but nevertheless preferentially hybridize to their respective target sequences. While being sufficiently complementary to hybridize to each other at ambient temperature. Exemplary probe pairs that meet the above criteria for Factor V and Factor V Leiden mutations are SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, Or any one of SEQ ID NO: 11. Exemplary probe pairs that meet the above criteria for mutations in the 3 ′ untranslated region of the prothrombin gene and the 3 ′ untranslated region of the prothrombin gene include SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or the sequence One of the numbers 19 is included.
[0031]
Probe pairs according to the present invention, such as those exemplified above or those meeting the criteria described above, are contemplated to include the target sequence and its variants (eg, the Factor V gene, or the Factor V Leiden mutation). And the portion that actually contains the Factor V Leiden mutation). In particular, a test sample suspected of containing the target sequence and a mutant form of the target sequence can be contacted with the probe pair in a single reaction vessel. Under suitable conditions known to those of skill in the art, probes hybridize to their respective target sequences, if present, to form probe target sequence hybrids. The hybrid formed between the probe and the target sequence can then be detected as an indicator of the presence of the target sequence in the test sample.
[0032]
In some cases, such as when the target sequence is not present in sufficient amounts for detection, the primer and amplification reagent may be added to the test sample before or simultaneously with contacting the probe with the test sample. . Depending on the nature of the probes and the target sequences to which they hybridize, often a set of amplification primers can be selected to amplify both target sequences. In particular, the probe can be selected such that the probe amplifies the target sequence in such a way that the mutation of the second target sequence, if present, is also included in the amplification product. For example, primers capable of amplifying a sequence comprising Factor V and a Factor V Leiden mutation include SEQ ID NO: 3 and SEQ ID NO: 4. Further, primers capable of amplifying mutations in the 3 ′ untranslated region of the prothrombin gene and the 3 ′ untranslated region of the prothrombin gene include SEQ ID NO: 14 and SEQ ID NO: 15.
[0033]
(Example)
Example 1
Array
A. Leiden Factor V Sequence: The following example demonstrates the detection of wild-type and mutant Leiden Factor V using DNA oligomer primers and the probes provided herein. These DNA primers and probes are identified as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. A portion of a representative sequence of wild-type Leiden Factor V is referred to herein as SEQ ID NO: 1, and a portion of a similar representative sequence of mutant Leiden Factor V is referred to herein as SEQ ID NO: 2. SEQ ID NO: 2 differs from SEQ ID NO: 1 only in one base which is a point mutation that distinguishes mutant Leiden Factor V from wild type. SEQ ID NO: 3 and SEQ ID NO: 4 are specific for regions found in both wild-type and mutant Leiden Factor V. SEQ ID NO: 5 is specific for a region found only in wild-type Leiden Factor V. SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 are specific for a region found only in mutant Leiden Factor V.
[0034]
In the examples below, SEQ ID NO: 3 and SEQ ID NO: 4 are used as amplification primers for both wild type and mutant Leiden Factor V. SEQ ID NO: 5 is used as an internal hybridization probe for wild-type Leiden Factor V amplification product. SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 are used as internal hybridization probes for mutant Leiden Factor V amplification products.
[0035]
B. Prothrombin Sequence: The following example demonstrates the detection of wild-type and mutant proton bin genes using DNA oligomer primers and the probes provided herein. These DNA primers and probes are identified as SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20. A portion of a representative sequence of the wild-type proton bin gene is referred to herein as SEQ ID NO: 12, and a portion of a similar representative sequence of the mutant proton bin gene is referred to herein as SEQ ID NO: 13. SEQ ID NO: 13 differs from SEQ ID NO: 12 only in one base, which is a point mutation that distinguishes the mutant proton bin gene from wild type. SEQ ID NO: 14 and SEQ ID NO: 15 are specific for regions found in both wild-type and mutant proton bin genes. SEQ ID NO: 16 is specific for a region found only in the wild-type proton bin gene. SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 are specific for regions found only in the wild-type proton bin gene.
[0036]
In the examples below, SEQ ID NO: 14 and SEQ ID NO: 15 are used as amplification primers for both wild-type and mutant proton bin genes. SEQ ID NO: 16 is used as an internal hybridization probe for the mutant proton bin gene amplification product. SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 are used as internal hybridization probes for the wild-type proton bin gene amplification product.
[0037]
Example 2
Preparation of primers and probes
A. Wild type and mutant Leiden Factor V primers: Primers were designed to detect both wild type and mutant Leiden Factor V target sequences by oligonucleotide hybridization PCR. These primers were SEQ ID NO: 3 and SEQ ID NO: 4. Primer sequences were synthesized using standard oligonucleotide synthesis techniques. The primer of SEQ ID NO: 4 was haptenated with a carbazole at the 5 'end using standard cyanoethyl phosphoramidite coupling chemistry as described in US Pat. No. 5,424,414, incorporated herein by reference. did.
[0038]
B. Wild-type and mutant Leiden Factor V probes: Probes were designed to hybridize to the amplified target sequence of wild-type or mutant Leiden Factor V by oligonucleotide hybridization. These probes have SEQ ID NO: 5 for wild-type Leiden Factor V and SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 for mutant Leiden Factor V. there were. Probe sequences were synthesized using standard oligonucleotide synthesis methods. The wild-type probe of SEQ ID NO: 5 was haptenized by adamantane at the 5 'end, then treated with 10 thymidine and blocked by phosphate at the 3' end. The SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 mutant probes are haptenized by dansyl at the 5 ′ end and then treated with 10 thymidine to 3 ′ The ends were blocked by phosphoric acid. All syntheses used standard cyanoethyl phosphoramidite coupling chemistry as described in US Pat. No. 5,464,746, which is incorporated herein by reference.
[0039]
C. Wild-type and mutant prothrombin primers: Primers were designed to detect target sequences of both wild-type and mutant prothrombin genes by oligonucleotide hybridization PCR. These primers were SEQ ID NO: 14 and SEQ ID NO: 15. Primer sequences were synthesized using standard oligonucleotide synthesis techniques. The primer of SEQ ID NO: 14 was prepared using standard cyanoethyl phosphoramidite coupling chemistry as described in US Pat. No. 5,424,414, incorporated herein by reference, to form a carbazole or adamantane at the 5 'end. Hapten by either. The primer of SEQ ID NO: 15 was haptenized with adamantane at the 5 'end by the same method.
[0040]
D. Wild-type and mutant prothrombin probes: Probes were designed to hybridize to the amplified target sequence of the wild-type or mutant prothrombin gene by oligonucleotide hybridization. These probes were SEQ ID NO: 16 for the mutant prothrombin gene and SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 for the wild type prothrombin gene. Probe sequences were synthesized using standard oligonucleotide synthesis methods. The mutant probe of SEQ ID NO: 16 is haptenized by 2 carbazoles at the 3 ′ end or by 2 dansyl at the 5 ′ end, then treated with 10 thymidine and phosphorylated at the 3 ′ end. Cut off by The wild-type probes of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 were haptenized at the 5 'end with 2 adamantane, then treated with 10 thymidine and blocked at the 3' end with phosphate. Separately, the wild type probes of SEQ ID NO: 17 and SEQ ID NO: 20 were haptenized with 2 carbazole at the 3 ′ end. All syntheses used standard cyanoethyl phosphoramidite coupling chemistry as described in US Pat. No. 5,464,746, which is incorporated herein by reference.
[0041]
Example 3
Sample preparation
The wild-type, heterozygous and homozygous mutant Leiden Factor V and prothrombin DNA were purified by the manufacturer using the QIAamp® Blood Mini Kit (Qiagen, Inc., Valencia, Calif.) Nucleic acid extraction procedure and column method. Purified from 200 μl of each whole blood sample as described. The DNA samples were simultaneously wild-type, heterozygous or homozygous mutants for both genes. The genotype of all samples was confirmed by sequencing. The purified DNA was quantified by reading the absorbance at 260 nm using a spectrophotometer. Final concentrations of purified DNA ranged from 50 to 25 ng / μl.
[0042]
Example 4
Detection of mutant versus wild-type Leiden Factor V using offset probes
The probes used to detect single base pair mutations that distinguish the mutant from wild-type Leiden Factor V were sense and antisense strand probes, respectively, and thus each other except for a single base pair mismatch. Exact complement. Therefore they can also hybridize with each other but not with their specific target amplified DNA. It was hypothesized that offsetting the probes slightly from each other would help minimize hybridization between them. This was tested by deleting two bases from the 5 'end of each probe.
[0043]
Duplicates of each of the purified wild type, heterozygous and homozygous mutant Leiden Factor V / prothrombin DNA samples were PCR amplified and sequenced along with the wild type probe of SEQ ID NO: 5 and the mutant probe of SEQ ID NO: 6. 3 and SEQ ID NO: 4. PCR was performed using 50 mM Bicine (N, N-bis [2-hydroxyethyl] glycine), pH 8.1, 150 mM potassium, 8% w / v glycerol, 0.001% bovine serum albumin (BSA), 0.1 mM EDTA and Performed in a buffer containing a final concentration of 0.02% sodium azide. Recombinant Thermus thermophilus polymerase was used at a concentration of 5 units / reaction with dNTPs (dATP, dGTP, dTTP and dCTP) each present at a final concentration of 150 μM. The primer of SEQ ID NO: 3 was used at a concentration of 120 nM, and the primer of SEQ ID NO: 4 (labeled with carbazole) was used at a concentration of 60 nM. The wild-type probe of SEQ ID NO: 5 was present at a concentration of 40 nM, and the mutant probe of SEQ ID NO: 6 was present at a concentration of 30 nM. For a sample volume of 20 μl, a final concentration of 3.25 mM manganese chloride was used in a total reaction volume of 0.2 ml.
[0044]
The reaction mixture was amplified on a LCx® Thermal Cycler. The reaction mixture was first incubated at 97 ° C. for 4 minutes and then subjected to 45 cycles of PCR amplification at 94 ° C. for 30 seconds and 55 ° C. for 45 seconds. After subjecting the reaction mixture to thermal cycling, the mixture was held at 97 ° C. for 5 minutes and the temperature was reduced to 15 ° C. to perform probe oligo hybridization. The sample was kept at 12 ° C for at least 5 minutes, after which time the reaction products were analyzed and detected.
[0045]
Reaction products were detected on an Abbott LCx® system (available from Abbott Laboratories, Abbott Park, IL). Using a suspension of anti-carbazole-coated microparticles, an anti-adamantane antibody / alkaline phosphatase conjugate and an anti-dansyl antibody / β-galactosidase conjugate (all available from Abbott Laboratories, Abbott Park, Ill.) With LCx® Reaction products were captured and detected. The enzyme substrates used were 4-methyl-umbelliferyl phosphate (MUP) and 7-β-D-galactopyranosyloxycoumarin-4-acetic acid- (2-hydroxyethyl) amide (AUG), which was produced from the substrate. The rate of conversion to the product was measured and reported as counts / sec / sec (c / s / s).
[0046]
The results in Table 1 show that the wild-type probe can detect both homozygous wild-type and heterozygous DNA, but not the homozygous mutant DNA (all expected), and that the mutant probe was homozygous. This indicates that neither the zygotic mutant nor the heterozygous DNA can be detected.
[0047]
[Table 1]

[0048]
Two bases at the 5 ′ end were removed and additional bases were added to the 3 ′ end with a mismatch G for destabilization (SEQ ID NO: 7, A) or without mismatch (SEQ ID NO: 11, E), Alternatively, two more bases are deleted at the 5 'end, but 3, 4 or 5 bases are added to the 3' end (SEQ ID NOs: 8 (B), 9 (C) or 10 (D), respectively), Modifications to the mutant probe were made. The experimental procedure described above was repeated using each of these mutant probes in place of SEQ ID NO: 6 in a separate reaction with the wild type probe of SEQ ID NO: 5. The results are shown in Table 2 below.
[0049]
[Table 2]

[0050]
All mutant probes performed better than the previous SEQ ID NO: 6 mutant probe and were capable of detecting both heterozygous and homozygous mutant DNA. With the exception of mutant probe D, the mutant probe also showed good specificity because no wild-type DNA was detected. The wild-type probe continued to perform well, detecting both wild-type and heterozygous DNA, but not homozygous mutant DNA. The best signal was achieved with mutant probes C (SEQ ID NO: 9) and D (SEQ ID NO: 10). Thus, the best combination of probes for detecting wild-type or mutant Leiden Factor V DNA is a wild-type probe having a 2 base deletion at the 5 'end (SEQ ID NO: 5), and also a 5' end. Was to use mutant probe C (SEQ ID NO: 9) which had a deletion of 2 bases but contained 4 additional bases at the 3 'end. This effectively shifts the base pair mismatch between the wild-type and mutant Leiden Factor V sequences from the middle to near the 5 'end.
[0051]
Thus, in general, offset probes were able to specifically hybridize to their appropriate amplified target sequence.
[0052]
Example 5
Detection of mutant versus wild-type prothrombin
A. Use of Sense Strand Probe: Initial experiments to detect wild-type versus mutant polymorphisms on the prothrombin gene were performed in a separate reaction with a similar sense strand probe, one for the mutant gene (SEQ ID NO: 16). The test was performed using a probe, the other for the wild-type gene (SEQ ID NO: 20). In addition to the single base pair difference at the wild type versus mutant locus, the wild type probe simultaneously contained two additional base pairs at the 5 'end.
[0053]
Duplicates of purified heterozygous and homozygous mutant Leiden Factor V / prothrombin DNA samples were prepared in a Molecular Grade Water (5'-3 ', Inc., Boulder, CO) at 1: 100, 1: 1000 and 1: SEQ ID NO: 16 mutant probe or carbazole-labeled SEQ ID NO: 20 wild-type, diluted in 10,000 and PCR amplified using adamantane labeled SEQ ID NO: 14 and SEQ ID NO: 15 primers in separate reactions Detection was performed using a probe. PCR was performed using 50 mM Bicine (N, N-bis [2-hydroxyethyl] glycine), pH 8.1, 150 mM potassium, 8% w / v glycerol, 0.001% bovine serum albumin (BSA), 0.1 mM EDTA and Performed in a buffer containing a final concentration of 0.02% sodium azide. Recombinant Thermus thermophilus polymerase was used at a concentration of 5 units / reaction with dNTPs (dATP, dGTP, dTTP and dCTP) each present at a final concentration of 150 μM. Primers were used at a concentration of 250 nM each and probes were present at a concentration of 5 nM each. For a sample volume of 20 μl, a final concentration of 3.25 mM manganese chloride was used in a total reaction volume of 0.2 ml. A 20 ng / μl poly-A pair was also tested as a negative control.
[0054]
The reaction mixture was amplified on a LCx® Thermal Cycler. The reaction mixture was first incubated at 95 ° C. for 4 minutes, then at 56 ° C. for 30 seconds, then at 97 ° C. for 2 minutes, and then subjected to 40 cycles of PCR amplification at 95 ° C. for 40 seconds and then at 58 ° C. for 60 seconds. . After subjecting the reaction mixture to thermal cycling, the mixture was held at 72 ° C. for 5 minutes, then at 97 ° C. for 5 minutes, and the temperature was reduced to 15 ° C. for 2 minutes to perform probe oligo hybridization. The reaction products were then analyzed and the samples were kept at 4 ° C. until detection.
[0055]
Reaction products were detected on an Abbott LCx® system (available from Abbott Laboratories, Abbott Park, IL). Reaction products were captured and detected using a suspension of anti-carbazole coated microparticles and an anti-adamantane antibody / alkaline phosphatase conjugate (all available from Abbott Laboratories, Abbott Park, IL) with LCx®. The enzyme substrate used was 4-methyl-umbelliferyl phosphate (MUP) and the rate of conversion of the substrate to product was measured and reported as counts / sec / sec (c / s / s).
[0056]
As can be seen from Table 3 below, the mutant probe detects both heterozygous and homozygous mutant samples, while the wild-type probe reacts only with the heterozygous probe and not with the homozygous mutant. Nevertheless, both samples were still detected. Therefore, this combination of probes, both probes from the sense strand, failed to distinguish wild type from mutant polymorphisms in the prothrombin gene.
[0057]
[Table 3]

[0058]
B. Use of Sense vs. Antisense Probe: Since the probe from the sense strand could not distinguish wild type from the mutant allele of the prothrombin gene, the wild type probe was transferred to the antisense strand, which We investigated whether it would be easier to identify genes.
[0059]
This experiment was performed as described in Example 5 above, except that the carbazole-labeled SEQ ID NO: 17 wild-type probe was used instead of the SEQ ID NO: 20 probe, and the duplicates of the samples were tested at only a 1: 100 dilution. A. Performed as described in. The thermal cycling conditions were also changed as follows: the reaction mixture was first incubated at 97 ° C for 2 minutes and then subjected to 40 cycles of PCR amplification at 95 ° C for 40 seconds and 58 ° C for 60 seconds. After subjecting the reaction mixture to thermal cycling, the mixture was held at 97 ° C. for 5 minutes, and the temperature was reduced to 15 ° C. for 2 minutes to perform probe oligo hybridization. The reaction products were then analyzed and the samples were kept at 15 ° C. until detection.
[0060]
[Table 4]

[0061]
The results in Table 4 above show that the transfer of the wild-type probe to the antisense strand allows discrimination between wild-type and the resulting mutant allele, and that the wild-type probe is more Also show a much higher detection signal for heterozygous samples containing one wild-type allele. Mutant probes also retain the ability to detect both heterozygous and homozygous mutant samples. Furthermore, under these conditions, the mutant probe was prepared as described in Example 5. A. Produces a slightly higher signal for homozygous mutant samples containing two mutant alleles than for heterozygous samples containing only one mutant allele, as compared to those observed in .
[0062]
C. Use of offset probe: Example 5 above. B. In one example, discrimination between wild-type and mutant prothrombin was achieved using one probe on the sense strand and the other probe on the antisense strand, but the sample was tested only once to determine genotype. Advantageously, both probes can be combined in one reaction mixture so that they do not have to be. Since this experiment also used sense (mutant) / antisense (wild-type) probes in separate reactions, two tests had to be performed on each sample to determine the genotype of the samples. However, since sense / antisense probes are exact complements of each other except for single base pair mismatches, they may also hybridize to each other rather than to their specific target amplified DNA. The thermal cycling conditions were modified to help prevent this, and in addition, the strategy used for the Leiden Factor V probe to offset the probes from each other near the base pair mismatch was utilized.
[0063]
A probe for detecting a mutant allele of the proton bin gene (SEQ ID NO: 16) was prepared in Example 5. A. And B. And contained a base pair mismatch in the middle (base pair 6 of 13 base pairs). Three wild-type probes were tested with this mutant probe: wild-type probe F (SEQ ID NO: 19) was complementary to the mutant probe except for a single base pair mismatch; wild-type probe G (sequence No. 17) contained two additional base pairs at the 3 'end; and wild-type probe H (SEQ ID NO: 18) contained two additional base pairs at the 3' end and two bases from the 5 'end. To create a two base overhang at the 3 'end of each probe.
[0064]
Duplicates of each of the purified wild-type, heterozygous and homozygous mutant Leiden Factor V / prothrombin DNA samples were PCR amplified and the primers SEQ ID NO: 14 and SEQ ID NO: 15 and the As in Example 4, except that the mutant probe was used with each of the three wild-type probes described above (ie, each one of the mutant probe and the wild-type probe was used in the same reaction mixture). Was detected. The reaction conditions used were as described in Example 4 above, except that the primer and probe concentrations were as follows: The primer of SEQ ID NO: 14 (labeled with carbazole) was at a concentration of 200 nM. The primers of SEQ ID NO: 15 were used at a concentration of 150 nM, the mutant probe of SEQ ID NO: 16 was used at a concentration of 70 nM, and the primers of SEQ ID NO: 17 (G), 18 (H) or 19 (F) Any of the wild-type probes was present in each reaction mixture at a concentration of 45 nM. The results from this experiment are shown in Table 5 below.
[0065]
[Table 5]

[0066]
All wild-type / mutant probe pairs showed specific detection of the appropriate wild-type or mutant allele, and wild-type probes detected homozygous wild-type and heterozygous prothrombin samples, but homozygous suddenly Mutant proton bin DNA was not detected as positive. The mutant probe detected homozygous mutant and heterozygous prothrombin samples, but did not detect homozygous wild-type proton bin samples as positive. As expected, all probes detected the heterozygous sample in a dose-dependent manner, since the heterozygous sample contained one wild-type and one mutant allele. Therefore, all probes showed high specificity.
[0067]
The wild-type probe (F), which is perfectly complementary to the mutant probe (except for a single base pair mismatch), worked, but produced the lowest signal for the homozygous wild-type sample. The wild-type probe that showed the best signal in detecting the wild-type allele was mutant probe H (SEQ ID NO: 18), a combination of probes that produced a two base pair overhang at both ends. This probe combination also showed the best balance (equivalence) between mutant and wild-type positive signals.
[0068]
Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made in such embodiments without departing from the spirit and scope of the invention.

Claims (18)

A method for detecting at least one of two related target nucleic acid sequences in a test sample, comprising:
a) contacting the test sample with the first and second probes, wherein:
(I) the probes are completely complementary to each other except for at least one mismatch,
(Ii) the first probe is more complementary to the sense strand of the first target sequence than to the second probe;
(Iii) the second probe is more complementary to the antisense strand of the second target sequence than to the first probe, and (iv) the probes hybridize to each other at ambient temperature b) at least the first or second Hybridizing the two probes to the first or second target sequence to form at least one target sequence / probe hybrid; and c) as an indicator of the presence of at least one target sequence in the test sample. Detecting the hybrid. 2. The method of claim 1, wherein the second target sequence is a mutated form of the first target sequence. 3. The method of claim 2, wherein the variant form of the first target sequence comprises a single nucleotide polymorphism. 4. The method of claim 3, wherein the probe is a member of the group consisting of SEQ ID NO: 5 and SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. 4. The method of claim 3, wherein the probe is a member of the group consisting of SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20. Applying a primer set and an amplification reagent to the test sample, and amplifying a target sequence in the test sample before or simultaneously with contacting the test sample with the first and second probes. 2. The method according to 1. The method according to claim 6, wherein the primer set is selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4; and SEQ ID NO: 14 and SEQ ID NO: 15. 2. The method of claim 1, wherein the first and second probes hybridize to each other such that the first probe has a 3 'overhang. 9. The method of claim 8, wherein the second probe has a 3 'overhang. 9. The method of claim 8, wherein the overhang is 1 to 5 nucleotides in length. A composition of matter comprising first and second probes, wherein:
(A) the probes are completely complementary to each other except for at least one mismatch,
(B) the first probe is more complementary to the sense strand of the target sequence than to the second probe;
A composition wherein (c) the second probe is more complementary to the antisense strand of the second target sequence than to the first probe, and (d) the probes hybridize to each other at ambient temperature. 12. The composition of claim 11, wherein the probes are individually 10 to 100 nucleotides in length. 13. The composition of claim 12, wherein the first and second probes hybridize to each other such that the first probe has a 3 'overhang. 14. The method of claim 13, wherein the second probe has a 3 'overhang. 14. The method of claim 13, wherein the overhang is 1 to 5 nucleotides in length. 12. The composition of claim 11, wherein the mismatch is a single nucleotide polymorphism. 4. The method of claim 3, wherein the probe is a member of the group consisting of SEQ ID NO: 5 and SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. 4. The method of claim 3, wherein the probe is a member of the group consisting of SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.