{"id":42789,"date":"2023-06-01T10:18:36","date_gmt":"2023-06-01T02:18:36","guid":{"rendered":"https:\/\/biopioneer.com.tw\/?p=42789"},"modified":"2023-06-27T11:30:12","modified_gmt":"2023-06-27T03:30:12","slug":"nanotaq-hot-start-dna-polymerase-bio-helix-%e8%b2%a8%e8%99%9fdp001-0100","status":"publish","type":"post","link":"http:\/\/biopioneer.com.tw\/?p=42789","title":{"rendered":"\u71b1\u555f\u52d5(Hot-Start ) DNA \u805a\u5408\u9176 | nanoTaq Hot-Start DNA Polymerase Bio-Helix \u8ca8\u865fDP001-0100"},"content":{"rendered":"

\u71b1\u555f\u52d5(Hot-Start ) DNA \u805a\u5408\u9176 nanoTaq Hot-Start DNA Polymerase| Bio-Helix \u8ca8\u865fDP001-0100<\/strong><\/a><\/span><\/h4>\n

nano Taq Hot-Start DNA Polymerase<\/p>\n

\u7522\u54c1\u63cf\u8ff0:<\/strong><\/span><\/p>\n

\u71b1\u555f\u52d5\u805a\u5408\u9176\u93c8\u5f0f\u53cd\u61c9\uff08Hot start PCR\uff09\u662f\u4e00\u7a2e\u6539\u826f\u7684\u805a\u5408\u9176\u93c8\u5f0f\u53cd\u61c9\u65b9\u6cd5\uff0c\u4e3b\u8981\u7528\u4f86\u907f\u514d\u64cd\u4f5c\u904e\u7a0b\u4e2d\u7522\u751f\u975e\u7279\u7570\u6027\u5e8f\u5217\u7684\u64f4\u589e\u3002Bio-Helix nanoTaq DNA \u805a\u5408\u9176(Bio-Helix nanoTaq<\/strong> DNA Polymerase , DP001-0100) \u70ba“\u589e\u5f37\u578b”<\/strong><\/span>\u71b1\u555f\u52d5\u9176 DNA \u805a\u5408\u9176\u4e4b\u4e00\uff0c\u70ba\u60a8\u7684\u7814\u7a76\u76ee\u6a19\u63d0\u4f9b\u4fbf\u5229\u548c\u53ef\u9760\u6027\u3002 nanoTaq \u63a1\u7528“\u7d0d\u7c73\u6280\u8853\u8907\u5408\u7269\u8a2d\u8a08”<\/span><\/strong>\uff0c\u662f\u4e00\u9805\u5275\u65b0\u5275\u9020\uff0c\u4e0d\u540c\u65bc\u50b3\u7d71\u7684\u71b1\u555f\u52d5\u9176\u88fd\u9020\u65b9\u6cd5\u3002 nanoTaq \u7d93\u9a57\u8b49\u7684\u529f\u80fd\u5305\u62ec\u5728\u5ba4\u6eab\u4e0b\u4f7f\u7528\u8207\u50b3\u7d71 Taq DNA \u805a\u5408\u9176\u76f8\u540c\u7684\u65b9\u6848\u548c\u5faa\u74b0\u689d\u4ef6\u9032\u884c\u53cd\u61c9\uff0c\u6e1b\u5c11\u975e\u7279\u7570\u6027\u5f15\u7269annealing\uff0c\u63d0\u9ad8PCR\u7522\u7269\u7522\u91cf\uff0c\u4e26\u53ef\u7528\u65bc\u9ad8\u9054 5kb \u7684 PCR \u7522\u7269\u3002<\/p>\n

    \n
  1. \u5c08\u4e00\u6027\u9ad8\uff0cPCR\u7522\u54c1\u53ef\u9ad8\u65bc5kb<\/span><\/li>\n
  2. \u9748\u654f\u5ea6\u9ad8\uff0c\u7528\u65bc GC >50% \u7684\u76ee\u6a19<\/span><\/li>\n
  3. \u5c0dhumic acid\u5e72\u64fe\u5177\u6709\u6700\u4f73\u8010\u53d7\u6027\u3002Hot-Start\u6700\u4f73\u9078\u64c7<\/span><\/li>\n<\/ol>\n

    \"\u7db2\u7ad9\u898f\u683c\u5c0f\u5716_\u5de5\u4f5c\u5340\u57df<\/a><\/p>\n

    <\/p>\n

    nanoTaq DNA polymerase provides both specificity and yield.<\/strong><\/p>\n\n\n\n\n\n\n\n
    A<\/strong><\/td>\nnanoTaq<\/strong><\/td>\n<\/tr>\n
    B<\/strong><\/td>\nThermo DreamTaq<\/td>\n<\/tr>\n
    C<\/strong><\/td>\nThermo DreamTaq HS<\/td>\n<\/tr>\n
    D<\/strong><\/td>\nRoche KAPA2G HS<\/td>\n<\/tr>\n
    E<\/strong><\/td>\nInvitrogen Platinum HS<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

    Figure 1. Comparison of non-specific amplifying effects to major suppliers. (\u9ad8\u5c08\u6027\u8868\u73fe)<\/span><\/strong><\/span>
    \nAll amplifications were performed in accordance with manufacturer\u2019s instructions to amplify 320 to 941 bp amplicons from human genomic DNA. nanoTaq provided both higher specificity and yield products with least band shifting compared to other commercial antibody-mediated hot start polymerase.<\/p>\n

    <\/p>\n

    Figure 2. Sensitivity and reliable amplification from low amounts of input DNA.(\u9748\u654f\u5ea6\u9ad8)<\/span>
    \nAmplification of a 803 bp fragment from 3; 0.3; 0.2; 0.1; 0.03; 0(no template control) ng of human genomic DNA were amplified in 20\u03bcL PCR reactions using nanoTaq DNA Polymerase.<\/p>\n

     <\/p>\n

    <\/p>\n

    Figure 3. Efficient amplification of DNA sequences with a range of GC content. (\u6709\u6548\u7528\u65bc GC >50% \u7684\u76ee\u6a19DNA)<\/span><\/p>\n

    A series of DNA fragments of increasing GC content were amplified from human gDNA. nanoTaq Hot-Start DNA Polymerase was used for targets with >50% GC.
    \n*Thermo DreamTaq,\u00a0Thermo DreamTaq HS and\u00a0Invitrogen Platinum HS are\u00a0registered trademarks of Thermo ScientificTM PierceTM.\u00a0Roche KAPA2G HS is a registered \u00a0trademark of Merck.\u00a0The trademark\u00a0holders\u00a0are\u00a0not affiliated with Bio-Helix Co., Ltd. and do\u00a0not endorse these products.<\/p>\n

     <\/p>\n

    <\/p>\n

    A. Thermo DreamTaq, Cat. EP0701
    \nB. Thermo DreamTaq HS, Cat. EP1701
    \nC. Roche KAPA2G HS, Cat. KK5523
    \nD. Invitrogen Platinum HS, Cat. 10966-018<\/p>\n

    Figure 4. Inhibitor resistance on nanoTaq DNA polymerase<\/span><\/strong><\/span>
    \nA 280 bp human genomic DNA target was conducted to compare with four leading competing DNA polymerases. All amplifications were performed in accordance with manufacturer\u2019s instructions where the mixtures contain:
    \n1. no inhibitor control,
    \n2. bile salts with final concentration of 0.5 mg\/mL,
    \n3. heparin with final concentration of 0.5 unit\/mL,
    \n4. sodium citrate with final concentration of 150 \u03bcg\/mL,
    \n5. hemin with final concentration of 0.1 \u03bcg\/mL and
    \n6. humic acid with final concentration of 1 \u03bcg\/mL.
    \nThe results show that nanoTaq has the best tolerance with the humic acid interference, when compared with major suppliers of the hot-start DNA polymerase.<\/p>\n

     <\/p>\n

    <\/p>\n

    Figure 5. Amplification of long target DNA fragments<\/span><\/strong>
    \nIt is generally known that Taq DNA polymerase exhibits difficulty amplifying DNA fragment sizes larger than 1 kb. This is probably due to the polymerase\u2019s error-prone amplification process which reduces the efficiency in amplifying longer target sizes. However, we have shown that\u00a0nanoTaq is capable of amplifying Lane M: 1Kb plus DNA Ladder RTU (DM015-R500), Lane A: 5 kb, Lane B: 3 kb and Lane C: 0.5 kb Lambda DNA fragment sizes under the 3-min extension time at 72\u00b0C. Therefore, nanoTaq shows the optimal performance in applications with long PCR fragments.<\/p>\n

    \u7522\u54c1\u7279\u5fb5:<\/strong><\/span><\/p>\n

    \u25ba High tolerance aganist various reaction inhibitors
    \n\u25ba\u00a0Reduce nonspecific primer annealing
    \n\u25ba Plays in efficient PCR amplification of\u00a0GC-rich sequences
    \n\u25ba Using the same protocol and cycling conditions as\u00a0conventional Taq DNA polymerases<\/p>\n

    \u7522\u54c1\u53cd\u61c9\u8a2d\u7f6e:<\/strong><\/span><\/p>\n

    1. For each 20 \u03bcl reaction, assemble the following in a 0.2 ml PCR tube on ice just prior to use:<\/p>\n\n\n\n\n\n\n\n\n\n\n\n
    <\/td>\nVolume<\/td>\nFinal Conc.<\/td>\n<\/tr>\n
    DNA template<\/td>\n– \u03bcl<\/td>\n3 ng<\/td>\n<\/tr>\n
    Forward primer, 5-10 \u03bcM<\/td>\n– \u03bcl<\/td>\n0.1-0.5 \u03bcM<\/td>\n<\/tr>\n
    Reverse primer, 5-10 \u03bcM<\/td>\n– \u03bcl<\/td>\n0.1-0.5 \u03bcM<\/td>\n<\/tr>\n
    dNTP Mix (10 mM each dATP, dCTP, dGTP, dTTP)<\/td>\n– \u03bcl<\/td>\n200 \u00b5M<\/td>\n<\/tr>\n
    10X PCR bu\ufb00er<\/td>\n2 \u03bcl<\/td>\n–<\/td>\n<\/tr>\n
    nanoTaq Hot-Start DNA Polymerase<\/td>\n-1 \u03bcl<\/td>\n–<\/td>\n<\/tr>\n
    PCR Grade Water<\/td>\nAdd to 20 \u03bcl<\/td>\n–<\/td>\n<\/tr>\n
    Total volume<\/td>\n20 \u03bcl<\/td>\n–<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

    2. Mix gently. If necessary, centrifuge briefly and cap tubes.<\/p>\n

    3. Place them into Thermocycler and process for 30-35 cycles as follows:<\/p>\n\n\n\n\n\n\n\n
    Initial Denaturation<\/td>\n\u00a03 min at 95\u00b0C<\/td>\n<\/td>\n<\/tr>\n
    Denaturation<\/td>\n30 sec<\/td>\n30-35 cycles<\/td>\n<\/tr>\n
    Annealing<\/td>\n30 sec at the proper annealing temperature<\/td>\n<\/tr>\n
    Extension<\/td>\n1 min at 72\u00b0C<\/td>\n<\/tr>\n
    Final extension<\/td>\n5 min at 72\u00b0C<\/td>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

    Note: Optimal conditions for amplification will vary depending on the primers and thermal cycler used. It may be necessary to optimize the system for individual primers, template, and thermal cycler.<\/p>\n

    * Template:
    \nPurified high quality DNA is needed for a success PCR reaction. The final concentration of cDNA template please refer to \u201cReaction Setup\u201d.<\/p>\n

    * Storage Bu\ufb00er:
    \nThe enzyme is supplied in a storage bu\ufb00er consisting of 20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM DTT, 50% (v\/v) glycerol, and 1% Triton X-100.<\/p>\n

    * Unit Definition:
    \nOne unit is defined as the amount of enzyme that will catalyze the incorporation of 10 nmol of dNTP into acid- insoluble form in 30 min at 74\u00b0C in a reaction containing 25 mM TAPS (tris-[hydroxymethyl]-methyl-amino- propane-sulfonic acid, sodium salt), pH 9.3 at 25\u00b0C, 50 mM KCl, 2 mM MgCl2<\/sub>, 1 mM \u03b2-mercaptoethanol, 0.2 mM dATP, dGTP, and dTTP, 0.1 \u03bcM [\u03b1-32P] dCTP, and activated salmon sperm DNA.<\/p>\n

    \u3010\u907a\u50b3\u5de5\u7a0bDNA\u5206\u5b50\u6280\u8853<\/a>\u3011<\/strong><\/span><\/p>\n

    \u805a\u5408\u9176\u93c8\u53cd\u61c9 (Polymerase Chain Reaction, PCR)\/DNA\u51dd\u81a0\u96fb\u6cf3<\/span><\/strong><\/p>\n

    PCR\u53cd\u61c9->\u74ca\u8102\u81a0\u9ad4\u7c89\u672b<\/a><\/span>\u914d\u88fd->DNA\u51dd\u81a0\u96fb\u6cf3\uff08agarose gel electrophoresis\uff09->\u6838\u9178\u67d3<\/a>\u8272<\/span>-><\/span><\/span><\/strong>\u5728\u96fb\u6cf3\u5b8c\u6210\u5f8c\u85c9\u7531\u85cd\u5149\u89c0\u5bdf\u7bb1<\/a><\/span>\u6fc0\u767c<\/strong>\u770b\u5230\u4e0d\u540c\u5927\u5c0f\u7684DNA\u7247\u6bb5\u3002<\/span><\/p>\n

    \"PCR\u53cd\u61c9\"<\/p>\n

    \u805a\u5408\u9176\u93c8\u53cd\u61c9 (Polymerase Chain Reaction, PCR)<\/span> <\/strong><\/span>\u662f\u4e00\u7a2e\u5206\u5b50\u6280\u8853(molecular technology)\uff0c\u7531\u8afe\u8c9d\u723e\u734e\u7372\u5f97\u8005 Kary Mullis \u5728 1980 \u5e74\u4ee3\u958b\u767c\uff0c\u53ef\u4ee5\u9ad4\u5916(in vivo) \u5feb\u901f\u4e14\u5ec9\u50f9\u5730\u64f4\u589e DNA \u7247\u6bb5(DNA fragments)(1)\u3002\u5b83\u5df2\u6210\u70ba\u907a\u50b3(\u00a0genetic )\u548c\u5206\u5b50\u7814\u7a76(molecular research)\u7684\u57fa\u672c\u5de5\u5177\uff0c\u56e0\u70ba DNA \u5be6\u9a57(DNA experimentation)\u901a\u5e38\u9700\u8981\u5927\u91cf\u7684\u76ee\u6a19 DNA (2)(3)\u3002\u4e00\u4e9b\u4f9d\u8cf4 PCR \u6280\u8853(PCR technology)\u7684\u61c9\u7528\u5305\u62ec DNA \u6e2c\u5e8f(DNA sequencing)\uff08\u4f8b\u5982\u4eba\u985e\u57fa\u56e0\u7d44\u8a08\u5283,Human Genome Project\uff09\u3001DNA \u6307\u7d0b\u8b58\u5225(DNA fingerprinting)\u3001\u6cd5\u91ab\u5b78(forensics)\u3001\u7d30\u83cc(bacteria)\u6216\u75c5\u6bd2(\u00a0viruses)\u6aa2\u6e2c\uff08\u5c24\u5176\u662f AIDS\uff09\u548c\u907a\u50b3\u6027\u75be\u75c5(hereditary disease)\u7684\u8a3a\u65b7 (4)(5)\u3002\u7531\u65bc\u80fd\u5920\u5f9e\u5c11\u91cf\u6838\u9178(nucleic acid)\u751f\u6210\u5927\u91cf DNA\uff0cPCR \u662f\u4e00\u7a2e\u975e\u5e38\u6709\u6548\u7684 DNA \u64f4\u589e\u65b9\u6cd5(amplify DNA )\uff0c\u56e0\u6b64\u6709\u6642\u88ab\u7a31\u70ba\u201c\u5206\u5b50\u5fa9\u5370(molecular photocopying)\u201d(2)(3)(6)\u3002<\/p>\n

    \u70ba\u4e86\u6aa2\u67e5\u662f\u5426\u64f4\u589e\u4e86\u6b63\u78ba\u7684\u76ee\u6a19 DNA \u7247\u6bb5\uff0c\u51dd\u81a0\u96fb\u6cf3(gel electrophoresis)\u662f\u6aa2\u67e5\u64f4\u589e\u7522\u7269(amplified products)\u5206\u5b50\u5927\u5c0f\uff08\u4ee5 bp \u70ba\u55ae\u4f4d\uff09\u7684\u5feb\u901f\u65b9\u6cd5\u3002\u6d41\u7a0b\u5982\u4e0b\uff1aPCR\u53cd\u61c9\u7522\u7269\uff0c\u7d93\u5fae\u91cf\u5206\u6ce8\u5668\u53d6\u5c11\u91cfLoading\u81f3\u914d\u88fd\u597d\u7684\u74ca\u8102\u81a0\u9ad4<\/a><\/span>\uff0c \u653e\u7f6e<\/strong>\u6c34\u5e73\u96fb\u6cf3\u69fd<\/a><\/span>\uff0c<\/strong>\u7d93\u51dd\u81a0\u96fb\u6cf3(gel electrophoresis)\u5f8c\uff0c<\/strong>\u96fb\u6cf3\u5b8c\u7684\u74ca\u8102\u81a0\u9ad4<\/a><\/span>, \u7d50\u5408DNA\u6838\u9178\u67d3\u5291<\/a><\/span>\uff0c<\/span><\/span><\/span><\/strong>\u5728\u85cd\u5149\u89c0\u5bdf\u7bb1<\/a><\/strong><\/span>\u6fc0\u767c\u4e0b\uff0c\u89c0\u5bdf\u4e0d\u540c\u5927\u5c0f\u7684DNA\u7247\u6bb5\u3002\u7d93\u7531\u64f4\u589e\u7684 PCR \u7522\u7269\u7684\u5927\u5c0f\u662f\u901a\u904e\u5c07\u5176\u8207DNA Ladder<\/a>\u00a0\u9032\u884c\u6bd4\u8f03\u4f86\u4f30\u8a08\u7684\uff0cDNA Ladder<\/a>\u662f\u4e00\u7a2e\u5206\u5b50\u91cf\u6a19\u8a18\uff0c\u5176\u4e2d\u5305\u542b\u5df2\u77e5\u5927\u5c0f\u7684 DNA \u7247\u6bb5\u3002<\/p>\n

    \"\"<\/p>\n

    \u8a31\u591a\u56e0\u7d20\u6703\u5e72\u64fe PCR \u53cd\u61c9\u3002\u6709\u4e9b\u6613\u65bc\u512a\u5316\uff08\u4f8b\u5982\u71b1\u5faa\u74b0\u689d\u4ef6\u548c Mg\u00a02+\u6fc3\u5ea6\uff09\uff0c\u800c\u53e6\u4e00\u4e9b\u5247\u64cd\u4f5c\u8d77\u4f86\u6bd4\u8f03\u68d8\u624b\uff0c\u9700\u8981\u7a4d\u7d2f\u7d93\u9a57\u624d\u80fd\u89e3\u6c7a\uff08\u4f8b\u5982\u5f15\u7269\u8a2d\u8a08\u548c PCR \u7de9\u885d\u6db2\u6210\u5206\/\u6dfb\u52a0\u5291\uff09\u3002\u4e00\u822c\u4f86\u8aaa\uff0cPCR \u4e2d\u7684\u91cd\u8981\u56e0\u7d20\u5305\u62ec DNA \u6a21\u677f\u3001DNA \u805a\u5408\u9176\u3001\u5f15\u7269\u8a2d\u8a08\u3001\u7de9\u885d\u6db2\u6210\u5206\u3001\u6dfb\u52a0\u5291\u548c\u6291\u88fd\u5291\u4ee5\u53ca\u71b1\u5faa\u74b0\u689d\u4ef6 (6)\u3002\u6216\u9078\u64c72XPCR\u6df7\u5408\u6db2<\/a>\u9032\u884c PCR \u53cd\u61c9\uff0c2X PCR \u6df7\u5408\u6db2 (2X PCR SuperMix, MB200-P100 )\u5177\u6700\u4f73\u4e4bMg ++\u3001dNTP\u548c\u91cd\u7d44Taq DNA\u805a\u5408\u9176\u7684\u6df7\u5408\u7269\u3002\u50c5\u9700\u52a0\u5165 template DNA\u3001primer \u6df7\u5408\u6db2\u5373\u53ef\u958b\u59cb\u9032\u884c PCR \u53cd\u61c9\u3002<\/p>\n

    \u74ca\u8102\u81a0\u9ad4\u7684\u88fd\u5099\uff1a\u53d6\u9069\u91cf\u74ca\u8102\u81a0\u9ad4\u7c89\u672b<\/a>\u653e\u5165\u7de9\u885d\u6db2\uff08\u5e38\u7528\u7684\u662fTBE\u7de9\u885d\u6db2\uff09\u4e2d\uff0c\u4ee5\u5fae\u6ce2\u52a0\u71b1\u81f3\u5b8c\u5168\u6eb6\u89e3\uff0c\u6df7\u5408\u5747\u52fb\u5f8c\uff0c\u51b7\u537b\u81f3\u7d0450\u2103\uff0c\u5012\u5165\u63d2\u6709\u9f52\u72c0\u6a21\u578b\u4e4b\u9444\u6a21\u4e2d\uff0c\u975c\u7f6e\u7b49\u5f85\u81a0\u9ad4\u51dd\u56fa\u5f8c\uff0c\u518d\u53d6\u4e0b\u9f52\u72c0\u6a21\u578b\u3002\u53d6\u51fa\u5df2\u5b8c\u5168\u51dd\u56fa\u7684\u81a0\u9ad4\uff0c\u7f6e\u65bc\u96fb\u6cf3\u69fd\u5167\uff0c\u4e26\u52a0\u5165\u7de9\u885d\u6db2\u81f3\u8986\u84cb\u81a0\u9ad4\u3002<\/p>\n

    \"PCR\u8a66\u5291_\u5de5\u4f5c\u5340\u57df<\/a><\/span><\/strong><\/p>\n

    PCR\u6df7\u5408\u6db2<\/a>\u00a0| 100bp-10kb DNA Ladder\u00a0<\/a>\u00a0|\u00a0\u74ca\u8102\u81a0\u9ad4\u7c89Agarose<\/a>\u00a0|\u00a0\u00a0DNA\u6838\u9178\u67d3\u5291<\/a><\/span><\/span><\/strong><\/p>\n

    \"BIO-HELIX\"\"BIO-HELIX<\/a>\"BIO-HELIX<\/a>\"BIO-HELIX<\/p>\n

     <\/p>\n

    \u805a\u5408\u9176\u934a\u5f0f\u53cd\u61c9 (PCR) \u662f Kary Mullis \u5728 1980 \u5e74\u4ee3\u958b\u767c\u7684\u9769\u547d\u6027\u65b9\u6cd5 [1]\uff0c\u70ba\u5206\u5b50\u751f\u7269\u5b78\u4e2d\u6700\u5f37\u5927\u7684\u6280\u8853\u4e4b\u4e00\u3002\u5e8f\u5217\u7279\u7570\u6027\u5f15\u5b50\u3001\u71b1\u7a69\u5b9a DNA \u805a\u5408\u9176\u548c\u71b1\u5faa\u74b0\u5c07 DNA \u6216 cDNA \u6a21\u677f\u4e2d\u7684\u7279\u5b9a\u5e8f\u5217\u5f9e\u5c11\u91cf\u64f4\u589e\u5230\u6578\u5343\u5230\u767e\u842c\u500d\u3002 20-40 \u6b21\u91cd\u8907\u7684\u52a0\u71b1\u548c\u51b7\u537b\u5faa\u74b0\uff08\u7a31\u70ba\u71b1\u5faa\u74b0\/thermal cycling\uff09\uff0c\u901a\u904e\u9176\u4fc3\u53cd\u61c9\u4fc3\u9032 DNA \u8907\u88fd(DNA replication)\u3002\u76ee\u6a19\u5e8f\u5217\u7684\u6578\u91cf\u96a8\u8457\u6bcf\u500b\u5faa\u74b0\u52a0\u500d\uff0c\u6307\u6578\u64f4\u589e\u300240 \u6b21\u91cd\u8907\u7684 PCR \u71b1\u5faa\u74b0\u5c07\u5f9e\u6a21\u677f\u7684\u55ae\u500b\u62f7\u8c9d\u751f\u6210 1,099,511,627,776 \u500b DNA \u62f7\u8c9d\u3002<\/p>\n

    In a nutshell, PCR is essentially a series of 20-40 repeated cycles of heating and cooling (called thermal cycling) that facilitate DNA replication through enzymatic reactions. The amount of target sequence doubles with each cycle which leads to an exponential amplification represented by 2(# of cycles)<\/sup>. Therefore, a PCR thermal cycling of 40 repeats will generate 1,099,511,627,776 copies of DNA from a single copy of the template. The function and purpose of each step in a PCR Reaction are discussed below\u00a0(Figure 1)<\/b>:<\/p>\n

    \"\"<\/p>\n

    Figure 1<\/b>\u00a0\u2013 How Polymerase Chain Reaction works: 1) Denaturation: the reaction is heated to 94\u201398 \u00b0C for 20\u201330 seconds to break the hydrogen bonds between the strands. 2) Annealing: the reaction temperature is lowered to 50-65 \u00b0C for 20-40 seconds to allow primers to anneal to the template strands. 3) Elongation: the temperature is increased (the optimal temperature is dependent on the DNA Polymerase used e.g. 72-78 \u00b0C for Taq Polymerase) to allow for the addition of dNTPs. The amount of target sequence doubles with each thermal cycle which leads to an exponential amplification represented by 2(# of cycles)<\/sup><\/p>\n

    \u8d77\u59cb\u53cd\u61c9( Initialization)<\/span><\/strong><\/span><\/p>\n

    \u5c07\u53cd\u61c9\u52a0\u71b1\u81f3 94\u201396\u00b0C\uff08\u5982\u679c\u4f7f\u7528\u6975\u8010\u71b1\u7684 DNA \u805a\u5408\u9176\uff0c\u5247\u52a0\u71b1\u81f3 98\u00b0C\uff092-10 \u5206\u9418\u3002\u8a72\u6b65\u9a5f\u6fc0\u6d3b\u53cd\u61c9\u4e2d\u7684 DNA \u805a\u5408\u9176\u548c\u8a66\u5291\uff0c\u4e26\u4f7f\u6df7\u5408\u7269\u4e2d\u7684\u5176\u4ed6\u6c61\u67d3\u7269\uff08\u5982\u679c\u6709\uff09\u8b8a\u6027\u3002\u5728\u83cc\u843d\u7be9\u9078\u7b49\u61c9\u7528\u4e2d\uff0c\u53ef\u76f4\u63a5\u4f7f\u7528\u5c11\u91cf\u7d30\u80de\u4f5c\u70ba\u6a21\u677f\u3002\u6b64\u521d\u59cb\u5316\u6b65\u9a5f\u5c07\u88c2\u89e3\u7d30\u80de\u4ee5\u91cb\u653e DNA \u4e26\u4f7f\u5176\u4ed6\u7d30\u80de\u86cb\u767d\u8cea\uff08\u5305\u62ec DNase\uff09\u8b8a\u6027\u3002\u5982\u679c\u5728 PCR \u671f\u9593\u4f7f\u7528\u5316\u5b78\u54c1\u6216\u6297\u9ad4\uff08\u4f8b\u5982 BlasTaq\u2122 HotStart DNA \u805a\u5408\u9176\uff09\uff0c\u5247\u5fc5\u9808\u57f7\u884c\u6b64\u6b65\u9a5f\u4ee5\u71b1\u6fc0\u6d3b\u9176\u3002<\/p>\n

    \u8b8a\u6027(Denaturation)<\/span><\/strong><\/span><\/p>\n

    \u5c07\u53cd\u61c9\u52a0\u71b1\u81f3 94\u201398\u00b0C \u4e26\u4fdd\u6301 20\u201330 \u79d2\u3002\u9019\u662f\u71b1\u5faa\u74b0(thermal cycling)\u7684\u7b2c\u4e00\u6b65\u3002\u96d9\u93c8 DNA(Double-stranded DNA) \u5728\u6b64\u6b65\u9a5f\u4e2d\u8b8a\u6027\uff0c\u56e0\u70ba\u96d9\u93c8\u4e4b\u9593\u7684\u6c2b\u9375(hydrogen bonds)\u56e0\u9ad8\u6eab\u800c\u65b7\u88c2\uff0c\u9019\u4e00\u904e\u7a0b\u4e5f\u7a31\u70ba DNA \u7194\u5316(DNA melting)\u3002
    \n\u93c8\u5408(Annealing)<\/strong><\/span><\/p>\n

    \u5f15\u5b50(Primers )\u662f\u53ef\u4ee5\u8207 DNA \u6a21\u677f(DNA template)\u7684\u7279\u5b9a\u5e8f\u5217(specific sequences)\u7d50\u5408\u4ee5\u6307\u5c0e DNA \u805a\u5408\u9176\u8907\u88fd\u7684\u5be1\u6838\u82f7\u9178(\u00a0oligonucleotides )\u3002\u70ba\u4f7f\u5f15\u7269(\u00a0primers )\u8207\u55ae\u93c8 DNA \u6a21\u677f\u00a0annealing\uff0c\u901a\u5e38\u5c07\u53cd\u61c9\u6eab\u5ea6\u964d\u81f3 50-65\u00b0C\uff0c\u6301\u7e8c 20-40 \u79d2\u3002Annealing\u7684\u6700\u4f73\u6eab\u5ea6\u53d6\u6c7a\u65bc\u5f15\u7269\u7684\u89e3\u93c8\u6eab\u5ea6 (T\u00a0m\u00a0)\uff1aDNA \u96d9\u93c8\u9ad4(DNA duplex)\u7684\u4e00\u534a\u89e3\u96e2\u6210\u55ae\u93c8\u7684\u6eab\u5ea6\u3002Annealing\u6eab\u5ea6\u8a2d\u7f6e\u904e\u9ad8\u5f15\u7269\u4e0d\u80fd\u8207\u6a21\u677fAnnealing\uff0cAnnealing\u6eab\u5ea6\u904e\u4f4e\u53ef\u80fd\u767c\u751f\u975e\u7279\u7570\u6027\u5f15\u7269\uff08\u5c0e\u81f4\u975e\u7279\u7570\u6027\u64f4\u589e\uff09\u3002\u56e0\u6b64\uff0c\u7406\u60f3\u7684Annealing\u6eab\u5ea6\u901a\u5e38\u6bd4 Tm \u4f4e 3-5\u00b0\u00a0C\u7684\u5f15\u7269((Primers )\uff0c\u8db3\u5920\u9ad8\u4ee5\u9032\u884c\u7279\u7570\u6027\u5f15\u5b50(Primers ) )Annealing\uff0c\u4f46\u53c8\u8db3\u5920\u4f4e\u4ee5\u5141\u8a31\u6709\u6548\u7684\u5f15\u5b50(Primers )\u3002\u53cd\u61c9\u6df7\u5408\u7269\u4e2d\u7684\u5f15\u5b50(Primers )\u6fc3\u5ea6\u901a\u5e38\u9060\u9ad8\u65bc DNA \u6a21\u677f\u7684\u6fc3\u5ea6\uff0c\u56e0\u6b64\u5f15\u5b50(Primers )-\u6a21\u677f\u96dc\u4ea4\u6bd4\u6a21\u677f\u93c8\u7684\u91cd\u65b0Annealing\u66f4\u53d7\u9752\u775e\u3002\u4e00\u65e6\u5f15\u5b50(Primers )\u8207\u6a21\u677fAnnealing\uff0cDNA \u805a\u5408\u9176\u5c31\u53ef\u4ee5\u958b\u59cb\u5c07 dNTP \u7d50\u5408\u5230\u6a21\u677f\u4e0a\u3002<\/p>\n

    \u5ef6\u4f38\/\u4f38\u9577(Extension\/Elongation)<\/span><\/strong><\/span><\/p>\n

    \u5728\u6b64\u6b65\u9a5f\u4e2d\uff0cDNA \u805a\u5408\u9176\u8207\u5f15\u5b50(Primers )\/\u6a21\u677f(template)\u8907\u5408\u7269\u7d50\u5408\uff0c\u4e26\u958b\u59cb\u5728\u5408\u6210\u93c8(synthesizing strand)\u4e0a\u4ee5 5′ \u5230 3′ \u7684\u65b9\u5411\u647b\u5165 dNTP\u3002\u9019\u6703\u7522\u751f\u4e00\u689d\u65b0\u5408\u6210\u7684\u8207\u6a21\u677f\u93c8(template strand)\u4e92\u88dc\u7684 DNA \u93c8\u3002\u5c0d\u65bc\u4e0d\u540c\u7684 DNA \u805a\u5408\u9176(\u00a0DNA polymerases)\uff0c\u5ef6\u4f38\/\u5ef6\u9577(extension\/elongation )\u7684\u6700\u4f73\u6eab\u5ea6\u4e0d\u540c\u3002Taq DNA \u805a\u5408\u9176(DNA polymerases)\uff08\u8010\u71b1\uff09\u7684\u7406\u60f3\u5de5\u4f5c\u6eab\u5ea6\u70ba 72-78\u00b0C\u3002\u5ef6\u4f38\u7684\u6301\u7e8c\u6642\u9593\u53d6\u6c7a\u65bc\u539f\u59cb\u6a21\u677f\uff08\u53c8\u540d\u64f4\u589e\u5b50\uff09\u7684\u9577\u5ea6\u548c DNA \u805a\u5408\u9176\u7684\u901f\u5ea6\u3002\u5178\u578b\u7684 DNA \u805a\u5408\u9176(\u00a0Typical DNA polymerases)\u5728\u5176\u6700\u4f73\u6eab\u5ea6\u7bc4\u570d\u5167\u4ee5 1-1.5kb\/min \u7684\u901f\u5ea6\u805a\u5408\u3002<\/p>\n

    \u6700\u7d42\u4f38\u9577\u7387(Final Elongation)<\/span><\/strong><\/p>\n

    \u5728\u6700\u5f8c\u4e00\u500b\u5ef6\u4f38\u5faa\u74b0\u4e2d\uff0c\u5c07\u53cd\u61c9\u6df7\u5408\u7269\u4fdd\u6301\u5728 72-78\u00b0C\uff08\u5927\u591a\u6578\u805a\u5408\u9176\u7684\u6700\u4f73\u5de5\u4f5c\u6eab\u5ea6\uff095-15 \u5206\u9418\u3002\u6b64\u6b65\u9a5f\u78ba\u4fdd\u4efb\u4f55\u5269\u9918\u7684\u55ae\u93c8 DNA \u5728\u6700\u5f8c\u4e00\u500b PCR \u5faa\u74b0\u5f8c\u5b8c\u5168\u5ef6\u4f38\u3002
    \n\u6700\u5f8c\u4fdd\u6301(Final Hold)<\/strong><\/span><\/p>\n

    \u5c07\u6eab\u5ea6\u964d\u4f4e\u81f3 4-15\u00b0C \u4e26\u6301\u7e8c\u7121\u9650\u9577\u7684\u6642\u9593\u4ee5\u77ed\u671f\u5132\u5b58\u53cd\u61c9\u6df7\u5408\u7269 (6)\u3002<\/p>\n","protected":false},"excerpt":{"rendered":"

    \u71b1\u555f\u52d5(Hot-Start ) DNA \u805a\u5408\u9176 nanoTaq Hot-Start DNA Polymeras […]<\/p>\n","protected":false},"author":3,"featured_media":42790,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":[],"categories":[5372],"tags":[5880],"_links":{"self":[{"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/posts\/42789"}],"collection":[{"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=42789"}],"version-history":[{"count":6,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/posts\/42789\/revisions"}],"predecessor-version":[{"id":43006,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/posts\/42789\/revisions\/43006"}],"wp:featuredmedia":[{"embeddable":true,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=\/wp\/v2\/media\/42790"}],"wp:attachment":[{"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=42789"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=42789"},{"taxonomy":"post_tag","embeddable":true,"href":"http:\/\/biopioneer.com.tw\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=42789"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}