鸡3种组织中热应激相关基因的表达谱芯片分析

Size: px

Start display at page:

Download "鸡3种组织中热应激相关基因的表达谱芯片分析"

娑穹舒
5 years ago
Views:

Hereditas (Beijing) 2018 年 3 月, 40(3): 227 236 www.chinagene.cn 综述低温变性下复合 PCR 技术及其应用梁卉 1,2, 陈国杰 3, 于燕 4, 熊礼宽 1,2 1. 518102 2. 518102 3. 450052 4.

1 Hereditas (Beijing) 2018 年 3 月, 40(3): 综述低温变性下复合 PCR 技术及其应用梁卉 1,2, 陈国杰 3, 于燕 4, 熊礼宽 1, 摘要 : 低温变性下复合 PCR(co-amplification at lower denaturation temperature-polymerase chain reaction, COLD- PCR) 是一种在高丰度野生型序列背景下选择性变性和扩增低丰度突变型序列的方法, 可将突变型序列富集 10~100 倍基于突变片段 Tm 值的改变和异源双链 DNA 分子的形成,COLD-PCR 可富集扩增片段中所有类型和位置的突变, 也可富集未知突变, 具有敏感特异精确廉价和易操作等优点 COLD-PCR 及其衍生方法被应用于肿瘤微生物产前筛查和动植物等领域, 对疾病的早期诊断病程和治疗监控药物选择预后判断和植物育种等均有积极的作用本文就 COLD-PCR 的原理关键技术衍生方法及其应用进行综述关键词 : 低温变性下复合 PCR; 精确的变性温度 ; 选择性富集 ; 低丰度突变 ; 检测阈值 Co-amplification at lower denaturation temperature-pcr: methodology and applications Hui Liang 1,2, Guojie Chen 3, Yan Yu 4, Likuan Xiong 1,2 1. Central Laboratory, Bao an Maternal and Child Health Hospital, Jinan University, Shenzhen , China 2. Shenzhen Key Laboratory of Birth Defects, Shenzhen , China 3. Department of Gastroenterology, First Affiliated Hospital of Zhengzhou University, Zhengzhou , China 4. Department of Obstetrics, Bao an Maternal and Child Health Hospital, Jinan University, Shenzhen , China Abstract: Co-amplification at lower denaturation temperature-polymerase chain reaction (COLD-PCR) is a novel form of PCR that selectively denatures and amplifies low-abundance mutations from mixtures of wild-type and mutation-containing sequences, enriching the mutation 10 to 100 folds. Due to the slightly altered melting temperature (Tm) of the dou- 收稿日期 : ; 修回日期 : 基金项目 : ( SZSM )( ZDSYS ) ( JD001) [Supported by Sanming Project of Medicine in Shenzhen-Brith Defects Prevention Research and Transformation Team (No. SZSM ), Shenzhen Key Laboratory of Birth Defects (No. ZDSYS and Science and Technology Plan Project of Baoan District (Nos , 2017JD001)] 作者简介 : E -mail: lianghui @126.com E -mail: chenguojie.hi@163.com 通讯作者 : E -mail: xionglk@sina.cn DOI: /j.yczz 网络出版时间 : 2018/2/8 11:31:42 URI:

2 228 Hereditas (Beijing) ble-stranded DNA and the formation of the mutation/wild-type heteroduplex DNA, COLD-PCR methods are sensitive, specific, accurate, cost-effective and easy to maneuver, and can enrich mutations of any type and at any position, even unknown mutations within amplicons. COLD-PCR and its improved methods are now applied in cancer, microorganisms, prenatal screening, animals and plants. They are extremely useful for early diagnosis, monitoring the prognosis of disease and the efficiency of the treatment, drug selection, prediction of prognosis, plant breeding and etc. In this review, we introduce the principles, key techniques, derived methods and applications of COLD-PCR. Keywords: co-amplification at lower denaturation temperature polymerase chain reaction (COLD-PCR); Tc; selectively amplifies; low-abundance mutation; detection threshold COLD-PCR COLD-PCR ( KRAS ) (0%~100%) [1] [4] COLD-PCR PCR ( 1) 1 COLD-PCR PCR(allele-specific amplification PCR, AS-PCR) COLD-PCR PCR PCR(wild-type blocking PCR, WTB- PCR) ( 2) Tc(critical denaturation temperature) (denaturing high performance liquid chromatography, dhplc) (melting temperature, Tm) PCR (inverse based Tm PCR- amplified restriction fragment length polymorphism, [3] iflp) [2] Tc COLD-PCR Tc (1) (G:C>A:T) PCR(co-amplification at lower denaturation temperature polymerase chain reaction, 0.2~1.5 [3] (2) DNA COLD-PCR) DNA DNA DNA Tm Tc DNA COLD -PCR DNA [3] Tc PCR Tm PCR Tm 0.5 Tm Tm [3] Tm 200 bp Tm 0.1~0.2

3 3 : PCR 229 PCR Fast COLD- Tc = Tm (1.0~2.0 ) COLD-PCR COLD- DNA Tc 1B) PCR(full COLD-PCR) COLD-PCR(fast COLD- PCR) Full COLD-PCR Full COLD-PCR fast COLD-PCR PCR COLD- fast COLD-PCR Tm PCR dgtp DNA Tc 58~ 62 datp ditp ddtp fast -PCR COLD ( Tm) 72 30~45 ( PCR 70 Fast COLD-PCR DNA [3] Tm [5] fast -COLD-PCR 1A) Full COLD-PCR DNA 图 1 Full COLD-PCR 和 fast COLD-PCR 原理图 Fig. 1 Schematic view of full COLD-PCR and fast COLD-PCR A Full COLD-PCR DNA95 70 DNA DNA DNA Tc DNA B Fast COLD-PCR PCR 95 Tc Tm

4 230 Hereditas (Beijing) COLD-PCR ±0.2 10~100 PCR Sanger COLD-PCR (high-resolution melting, HRM) PCR (next n- PCR ge Tc COLD-PCR eration sequencing, NGS) 10%~30% 10% 5%~15% 0.1% 2%~5% COLD-PCR 0.5%~3% 0.5%~ Tc 1.0% [3] 1%~2% [6] 0.2%~1.2% [7] 0.02%~1% [8] ( Tc COLD-PCR 1) Li [9] fast COLD- PCR nested fast COLD-PCR Sanger 0.01% 2.1 改良型完全富集 COLD-PCR (improved Kristensen [10] KRAS and complete enrichment COLD-PCR, ice-cold-pcr) COLD-PCR COLD-PCR Ice-COLD-PCR COLD-PCR Song [6] COLD-PCR/HRM 7% DMSO 0.2%~0.3% TP53 DMSO RS) DNA (1%~2%) 10 Hashida [11] COLD-PCR/HRM 0.01% (1) (2)(3)3 RS COLD-PCR EGFR T790M BstUⅠ Tc RS - full- COLD-PCR DNA ice-cold-pcr 30 s full 2 COLD-PCR COLD-PCR 5~8 min Ice- COLD-PCR Tc full COLD-PCR(3~5 ) fast COLD-PCR 表 1 COLD-PCR 的方法学比较 Table 1 Comparison of COLD-PCR methods PCR Full COLD-PCR Fast COLD-PCR Ice-COLD-PCR E-ice-COLD-PCR TT-COLD-PCR Tm 3~10 10~ ~16 6~17 RS/ ( 20 nmol/l ) LNA( GC 10 nmol/l) Tc ±2 ±2 ±2 5~10 2.5~3 2~8 min 30 s 30 s 2~8 min Sanger 10%~30% 3% 0.5% 1% 0.1% 3% ~10% 0.5%~1% 0.1% 0.01% 5%~10% 1~2% 2% 3% (HRM) PCR 0.1 % 0.2%~1.2% 2%~5% 0.02%~0.2% 0.01%~0.1% (NGS)

5 3 : PCR 231 (~10 ) ice-cold-pcr 5~10 1%(Sanger ) 0.1%( ) [12] COLD- COLD-PCR 10 PCR TT-fast- TT-full- TT-ice-COLD- KRAS ( Transgenomic, PCR PCR Omaha, NE) ice- COLD-PCR 8~17 6~8 12 COLD-PCR Tc DMSO Tm Tc TT-COLD-PCR How-Kit [4] ice-cold-pcr TT-COLD-PCR RS (locked nucleic acid, PCR LNA) E-ice-COLD-PCR(enhanced-ice- (10 ice-cold-pcr nmol/l) (115 ) COLD-PCR) E-ice-COLD-PCR (1) COLD-PCR 25 ng DNA TT-COLD-PCR Tc COLD-PCR 3 h (2) Tc Tc 5~10 COLD-PCR [4] (3) [13] (4) COLD-PCR [14] (5) E-ice-COLD-PCR Castellanos -Rizaldos [17] TT-fast-eCOLD-PCR (TT-fast- (20 nmol/l) WTB-PCR(1~100 µmol/l) (6) COLD-PCR in emulsion) PCR 0.01~0.05%( ) ( ) (7) LNA [4] TT-fast-eCOLD-PCR (8) DNA E-ice-COLD-PCR 3 PCR (formalin -fixed paraffin-embedded, FFPE) [15] ditp ddtp dgtp datp E-ice-COLD-PCR Tm LNA-DNA 1 2 fast- G-T Tm TT-COLD-PCR NGS (0.01%~0.1%) Sanger E -ice-cold-pcr LNA (0.1%~0.3%) [5] G-T E -ice-cold-pcr COLD-PCR/NGS(0.02%~1%) [8] 100% COLD-PCR [4] 3 COLD-PCR 2.2 温度耐受性 COLD-PCR(temperature-tolerant COLD-PCR, TT-COLD-PCR) Castellanos-Rizaldos [16] TT-COLD COLD-PCR 与肿瘤 COLD-PCR 与 KRAS 基因 PCR COLD-PCR Tc 2~3 COLD-PCR TT-COLD-PCR COLD-PCR KRAS PCR 0.3~0.5 7~15 30%~40%

6 232 Hereditas (Beijing) (colorectal cancer, CRC) KRAS curve analysis, FMCA) IDH1 COLD-PCR CRC KRAS 1% Boisselier [24] PCR/HRM 50% [18,19] 80% PCR/COLD-PCR/HRM COLD-PCR/COLD-PCR/ [10] KRAS EGFR HRM IDH1 COLD-PCR 25% 2% 0.25% COLD-PCR/COLD- [3] PCR/HRM COLD-PCR COLD -PCR/FMCA PCR COLD-PCR/FMCA ( FFPE ) Kanamori [25] IDH1 ( DNA(circulating IDH2 tumor DNA, ctdna)) DNA (15 fast COLD-PCR min) Liu [20] COLD-PCR min ctdna ctdna KRAS 75% COLD-PCR ctdna [21] COLD-PCR KRAS 0.01%~5% Sanger ctdna PCR/Sanger 10~30% COLD-PCR/Sanger ~5% ctdna Boisselier [26] COLD- [21] Sanger HRM PCR PCR ctdna PCR/ HRM 5%~10% KRAS COLD- IDH1 37.5% 70.6% COLD- PCR/HRM 3% [22] Pritchard PCR [19] fast COLD-PCR/HRM-cy5.5 COLD-PCR 1% COLD-PCR/ 0.5%~1% E-ice-COLD-PCR ARMS (0.1%~1%) [4] (40~45 min) FMCA (5 min) 60~ ctdna E-ice-COLD- 0.1~0.01% PCR IDH1 IDH COLD-PCR 与异柠檬酸脱氢酶 1(isocitrate dehydrogenase 1, IDH1) 基因 COLD-PCR 与其他肿瘤相关基因 COLD-PCR COLD -PCR Pang [23] Fast COLD-PCR/ COLD-PCR/(fluorescence specific melting restriction enzyme digestion, MARED)

7 3 : PCR 233 GNAS % COLD-PCR(modified COLD-PCR, mcold- (17/28) PCR) COLD-PCR GNAS1 (denaturing gradient gel electrophoresis, DGGE) [27] COLD-PCR mcold-pcr/dgge (congenital ( melanocytic nevus, CMN) NRAS BRAF ) COLD -PCR NRAS (22%~80%) Charbel [28] E-ice-COLD-PCR 2 CMN mcold-pcr NGS (>20 (10~20 cm)cmn cm)cmn 80.8% 8.8% NRAS 94.7% BRAF 1 - CMN NRAS 70% BRAF 30% NRAS CMN COLD-PCR COLD-PCR EGFR p53 BRAF( ) [15] NF2( Tc 2 ) [29] H-ras( ) [30] c-kit( ) [31] COLD-PCR COLD-PCR COLD-PCR [35] 3.2 COLD-PCR 与微生物感染 3.3 COLD-PCR 与产前筛查 DNA COLD-PCR (cell-free fetal DNA, cfdna) 5~20 COLD-PCR [36] cfdna 50 DNA 3%~20% COLD-PCR IU/mL(fast COLD-PCR)~100 IU/mL(full COLD-PCR) [32] (line probe assay, LiPA) COLD-PCR [33] COLD-PCR [36,37] Full COLD-PCR/Sanger β (COLD-PCR 148/215 LiPA 155/215) 1/ [38] Galbiati [39] COLD-PCR TWIST 18 bp Takahashi [34] COLD-PCR

8 234 Hereditas (Beijing) COLD-PCR/ CYP21 p.q318x PCR [40] DNA COLD- Galbiati [41] Full- fast- COLD-PCR β KRAS COLD -PCR COLD-PCR E-ice-COLD-PCR C OLD-PCR 1 5 Tc COLD-PCR 参考文献 (References): E-ice-COLD- PCR [1] Lu YW, Wang KH. Research progress on genetic heterogeneity between primary and paired metastatic colorectal 3.4 COLD-PCR 在其他方面的应用 cancer. Hereditas (Beijing), 2017, 39(6): ,. (C)., 2017, 39(6): (U) Tm 5 [2] Milbury CA, Li J, Makrigiorgos GM. PCR-based methods COLD-PCR DNA for the enrichment of minority alleles and mutations. Clin Castellanos-Tizaldos [42] fast COLD-PCR Chem, 2009, 55(4): [3] Li J, Wang LL, Mamon H, Kulke MH, Berbeco R, PCR(methylation-specific PCR, MS- PCR) Makrigiorgos GM. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity O6- - DNA fast COLD-MS-PCR DNA of genetic testing. Nat Med, 2008, 14(5): % MS-PCR 5% [4] How Kit A, Mazaleyrat N, Daunay A, Nielsen HM, Terris COLD-PCR Chen [43] B, Tost J. Sensitive detection of KRAS mutations using enhanced-ice-cold-pcr mutation enrichment and direct COLD-PCR/HRM PpAG PpTFL1 sequence identification. Hum Mutat, 2013, 34(11): [5] Castellanos-Rizaldos E, Richardson K, Lin R, Wu G, Makrigiorgos MG. Single-tube, highly parallel mutation 4 enrichment in cancer gene panels by use of temperature-tolerant COLD-PCR. Clin Chem, 2015, 61(1): COLD-PCR [6] Yuan F, Xu J, Ji LD, Fei LJ, Liu PP, Zhang LN. Application PCR of Tm-shift genotyping method in genetic studies. Hereditas (Beijing), 2012, 34(11): COLD-PCR DNA(mitochondrial DNA, mtdna) fast COLD-PCR (16%(5/31)) mtdna (Tm ) full -PCR COLD [45] [38,41] PCR COLD-PCR [44] COLD-PCR

9 3 : PCR 235,,,,,. - Tm Enrichment of mutations in multiple DNA sequences using shift., 2012, 34 COLD-PCR in emulsion. PLoS One, 2012, 7(12): e (11): [7] Castellanos-Rizaldos E, Paweletz C, Song C, Oxnard GR, [18] Zuo Z, Chen SS, Chandra PK, Galbincea JM, Soape M, Mamon H, Jänne PA, Makrigiorgos GM. Enhanced ratio of Doan S, Barkoh BA, Koeppen H, Medeiros LJ, Luthra R. signals enables digital mutation scanning for rare allele Application of COLD-PCR for improved detection of detection. J Mol Diagn, 2015, 17(3): KRAS mutations in clinical samples. Mod Pathol, 2009, [8] Milbury CA, Correll M, Quackenbush J, Rubio R, 22(8): Makrigiorgos GM. COLD-PCR enrichment of rare cancer [19] Pritchard CC, Akagi L, Reddy PL, Joseph L, Tait JF. mutations prior to targeted amplicon resequencing. Clin COLD-PCR enhanced melting curve analysis improves Chem, 2012, 58(3): diagnostic accuracy for KRAS mutations in colorectal carcinoma. BMC Clin Pathol, 2010, 10: 6. [9] Li J, Milbury CA, Li C, Makrigiorgos GM. Two- round coamplification at lower denaturation temperature-pcr [20] Liu PJ, Liang HY, Xue L, Yang C, Liu Y, Zhou K, Jiang (COLD-PCR)-based sanger sequencing identifies a novel XF. Potential clinical significance of plasma-based KRAS spectrum of low-level mutations in lung adenocarcinoma. mutation analysis using the COLD-PCR/TaqMan -MGB Hum Mutat, 2009, 30(11): probe genotyping method. Exp Ther Med, 2012, 4(1): [10] Kristensen LS, Daugaard IL, Christensen M, Hamilton-Dutoit S, Hager H, Hansen LL. Increased sensitivity [21] Carotenuto P, Roma C, Cozzolino S, Fenizia F, Rachiglio of KRAS mutation detection by high-resolution melting AM, Tatangelo F, Iannaccone A, Baron L, Botti G, analysis of COLD-PCR products. Hum Mutat, 2010, 31 Normanno N. Detection of KRAS mutations in colorectal (12): cancer with fast COLD-PCR. Int J Oncol, 2012, 40(2): [11] Hashida S, Soh J, Toyooka S, Tanaka T, Furukawa M, Shien K, Yamamoto H, Asano H, Tsukuda K, Hagiwara K, [22] Mancini I, Santucci C, Sestini R, Simi L, Pratesi N, Miyoshi S. Presence of the minor EGFR T790M mutation Cianchi F, Valanzano R, Pinzani P, Orlando C. The use of is associated with drug-sensitive EGFR mutations in lung COLD-PCR and high-resolution melting analysis improves the limit of detection of KRAS and BRAF mutations adenocarcinoma patients. Oncol Rep, 2014, 32(1): in colorectal cancer. J Mol Diagn, 2010, 12(5): [12] Milbury CA, Li J, Makrigiorgos GM. Ice-COLD-PCR enables rapid amplification and robust enrichment for low- [23] Pang B, Durso MB, Hamilton RL, Nikiforova MN. A nov- abundance unknown DNA mutations. Nucleic Acids Res, el COLD-PCR/FMCA assay enhances the detection of 2011, 39(1): e2. low-abundance IDH1 mutations in gliomas. Diagn Mol [13] Tost J. The clinical potential of enhanced-ice-cold-pcr. Pathol, 2013, 22(1): Expert Rev Mol Diagn, 2016, 16(3): [24] Boisselier B, Marie Y, Labussière M, Ciccarino P, [14] Mauger F, Daunay A, Deleuze JF, Tost J, How-Kit A. Desestret V, Wang XW, Capelle L, Delattre JY, Sanson M. Multiplexing of E-ice-COLD-PCR Assays for mutation COLD PCR HRM: a highly sensitive detection method for detection and identification. Clin Chem, 2016, 62(8): IDH1 mutations. Hum Mutat, 2010, 31(12): [15] How-Kit A, Lebbé C, Bousard A, Daunay A, Mazaleyrat N, [25] Kanamori M, Kikuchi A, Watanabe M, Shibahara I, Saito Daviaud C, Mourah S, Tost J. Ultrasensitive detection and R, Yamashita Y, Sonoda Y, Kumabe T, Kure S, Tominaga T. identification of BRAF V600 mutations in fresh frozen, Rapid and sensitive intraoperative detection of mutations FFPE, and plasma samples of melanoma patients by E-ice- in the isocitrate dehydrogenase 1 and 2 genes during surgery for glioma. J Neurosurg, 2014, 120(6): COLD-PCR. Anal Bioanal Chem, 2014, 406(22): [16] Castellanos-Rizaldos E, Liu PF, Milbury CA, Guha M, [26] Boisselier B, Gállego Pérez-Larraya J, Rossetto M, Brisci A, Cremonesi L, Ferrari M, Mamon H, Makrigiorgos GM. Temperature-tolerant COLD-PCR reduces M. Detection of IDH1 mutation in the plasma of patients Labussière M, Ciccarino P, Marie Y, Delattre JY, Sanson temperature stringency and enables robust mutation enrichment. Clin Chem, 2012, 58(7): [27] Delaney D, Diss TC, Presneau N, Hing S, Berisha F, with glioma. Neurology, 2012, 79(16): [17] Castellanos-Rizaldos E, Milbury CA, Makrigiorgos GM. Idowu BD, O'Donnell P, Skinner JA, Tirabosco R, Flana-

10 236 Hereditas (Beijing) gan AM. GNAS1 mutations occur more commonly than previously thought in intramuscular myxoma. Mod Pathol, 2009, 22(5): [28] Charbel C, Fontaine RH, Malouf GG, Picard A, Kadlub N, El-Murr N, How-Kit A, Su XP, Coulomb-L'Hermine A, Tost J, Mourah S, Aractingi S, Guégan S. NRAS mutation is the sole recurrent somatic mutation in large congenital melanocytic nevi. J Invest Dermatol, 2014, 134(4): [29] Paganini I, Mancini I, Baroncelli M, Arena G, Gensini F, Papi L, Sestini R. Application of COLD-PCR for improved detection of NF2 mosaic mutations. J Mol Diagn, 2014, 16(4): [30] Wang AX, Chang JW, Li CY, Liu K, Lin YL. H-ras mutation detection in bladder cancer by COLD-PCR analysis and direct sequencing. Urol Int, 2012, 88(3): [31] Gentilini F, Mantovani V, Turba ME. The use of COLD- PCR, DHPLC and GeneScanning for the highly sensitive detection of c-kit somatic mutations in canine mast cell tumours. Vet Comp Oncol, 2015, 13(3): [32] Liu C, Lin JP, Chen HJ, Shang HY, Jiang L, Chen J, Ye Y, Yang B, Ou QS. Detection of hepatitis B virus genotypic resistance mutations by coamplification at lower denaturation temperature-pcr coupled with sanger sequencing. J Clin Microbiol, 2014, 52(8): [33] Wong DKH, Tsoi O, Huang FY, Seto WK, Fung J, Lai CL, Yuen MF. Application of coamplification at lower denaturation temperature-pcr sequencing for early detection of antiviral drug resistance mutations of hepatitis B virus. J Clin Microbiol, 2014, 52(9): [34] Takahashi M, Masaki K, Mizuno A, Goto-Yamamoto N. Modified COLD-PCR for detection of minor microorganisms in wine samples during the fermentation. Food Microbiol, 2014, 39: [35] Wong DK, Fung J, Lai CL, Yuen MF. COLD-PCR for early detection of hepatitis B virus antiviral drug resistance mutations. Hong Kong Med J, 2015, 21(Suppl 7): S8 S10. [36] Ferro M, Macher HC, Noguerol P, Jimenez-Arriscado P, Molinero P, Guerrero JM, Rubio A. Non-invasive prenatal diagnosis of feto-maternal platelet incompatibility by cold high resolution melting analysis. Adv Exp Med Biol, 2016, 924: [37] Macher HC, Martinez-Broca MA, Rubio-Calvo A, Leon- Garcia C, Conde-Sanchez M, Costa A, Navarro E, Guerrero JM. Non-invasive prenatal diagnosis of multiple endocrine neoplasia type 2A using COLD-PCR combined with HRM genotyping analysis from maternal serum. PLoS One, 2012, 7(12): e [38] Galbiati S, Brisci A, Lalatta F, Seia M, Makrigiorgos GM, Ferrari M, Cremonesi L. Full COLD-PCR protocol for noninvasive prenatal diagnosis of genetic diseases. Clin Chem, 2011, 57(1): [39] Galbiati S, Stenirri S, Sbaiz L, Barberis M, Cremonesi L, Restagno G, Ferrari M. Further considerations concerning non-invasive prenatal diagnosis of craniosynostosis based on the identification of an 18 bp deletion in the TWIST1 gene by COLD-PCR. Clin Chem Lab Med, 2014, 52(7): e129 e130. [40] Du J, Zou X, Pan Y, Li SF, Lu GX. Non-invasive prenatal molecular detection of a fetal point mutation for congenital adrenal hyperplasia using co-amplification at lower denaturation temperature PCR. Chin Med J (Engl), 2010, 123(22): [41] Galbiati S, Monguzzi A, Damin F, Soriani N, Passiu M, Castellani C, Natacci F, Curcio C, Seia M, Lalatta F, Chiari M, Ferrari M, Cremonesi L. COLD-PCR and microarray: two independent highly sensitive approaches allowing the identification of fetal paternally inherited mutations in maternal plasma. J Med Genet, 2016, 53(7): [42] Castellanos-Rizaldos E, Milbury CA, Karatza E, Chen CC, Makrigiorgos GM, Merewood A. COLD-PCR amplification of bisulfite-converted DNA allows the enrichment and sequencing of rare un-methylated genomic regions. PLoS One, 2014, 9(4): e [43] Chen YH, Wilde HD. Mutation scanning of peach floral genes. BMC Plant Biol, 2011, 11: 96. [44] Li XZ. Enrichment of minority alleles in mixture by COLD-PCR[D]. Luoyang: Henan University of Science and Technology, PCR COLD [ ]. :, [DO [45] Meng XJ, Zhu JP, Gao M, Zhang S, Zhao FX, Zhang JJ, Liu XL, Wei QP, Tong Y, Zhang ML, Qu J, Guan MX. The analysis of mitochondrial DNA haplogroups and variants for Leber's hereditary optic neuropathy in Chinese families carrying the m.14484t>c mutation. Hereditas (Beijing), 2014, 36(4): ,,,,,,,,,,,. m.14484t>c Leber s., 2014, 36(4): ( 责任编委 : 卢大儒 )

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr PCR 龙平, 崔占虎, 李虔全, 徐建平, 张春红, 周立社 * *, 李旻辉 ( 包头医学院, 内蒙古包头 014060) DNA 30 28 DNA trnl-trnf PCR PCR 136 bp 323 bp 10% PCR PCR Astragalus membranaceus Fisch. Bge. var. mongho- licus Bge. Hsiao A. membranaceus