Pyrosequencing™ technology at elevated temperature

被引：6

作者：

Eriksson, J ^{[1
]}

Gharizadeh, B ^{[1
]}

Nordström, T ^{[1
]}

Nyrén, P ^{[1
]}

机构：

[1] Royal Inst Technol, SCFAB, Dept Biotechnol, SE-10691 Stockholm, Sweden

来源：

ELECTROPHORESIS | 2004年 / 25卷 / 01期

关键词：

DNA structures; luciferase; pyrosequencing;

D O I：

10.1002/elps.200305708

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

To date, the Pyrosequencing(TM) technology has been performed at 28degreesC due to the low thermostability of the firefly luciferase. In this study, firefly luciferase was stabilized in the presence of glycine betaine, allowing DNA sequencing at 37degreesC. By increasing the temperature to 37degreesC, false signals due to primer-dimers and loop-structures were decreased significantly. In addition, a combination of (i) replacing the natural dGTP with 7'deaza-dGTP in the polymerase chain reaction (PCR), (ii) 1.6 m glycine betaine, and (iii) an increase of the temperature to 37degreesC enabled us to sequence a DNA template with the initial sequence 3'-ATGGCCCGGGGGGGAGCTCCA . . . 5'. Furthermore, we describe a method to analyze if a primer forms a primer-dimer with extendable 3'-ends.

引用

页码：20 / 27

页数：8

共 16 条

[1]

BARR PJ, 1986, BIOTECHNIQUES, V4, P428

[2]

CASASFINET JR, 1987, J BIOL CHEM, V262, P8574

[3]

Dawson M C., 1986, DATA BIOCH RES

[4] Overexpression, rapid isolation, and biochemical characterization of Escherichia coli single-stranded DNA-binding protein [J].

Ehn, M ;

Nilsson, P ;

Uhlén, M ;

Hober, S .

PROTEIN EXPRESSION AND PURIFICATION, 2001, 22 (01) :120-127

[5]

Ehn M, 2002, ELECTROPHORESIS, V23, P3289, DOI 10.1002/1522-2683(200210)23:19<3289::AID-ELPS3289>3.0.CO

[6]

2-#

[7] Method enabling firefly luciferase-based bioluminometric assays at elevated temperatures [J].

Eriksson, J ;

Nordström, T ;

Nyrén, P .

ANALYTICAL BIOCHEMISTRY, 2003, 314 (01) :158-161

[8] Betaine improves the PCR amplification of GC-rich DNA sequences [J].

Henke, W ;

Herdel, K ;

Jung, K ;

Schnorr, D ;

Loening, SA .

NUCLEIC ACIDS RESEARCH, 1997, 25 (19) :3957-3958

[9] Production, purification, and luminometric analysis of recombinant Saccharomyces cerevisiae MET3 adenosine triphosphate sulfurylase expressed in Escherichia coli [J].

Karamohamed, S ;

Nilsson, J ;

Nourizad, K ;

Ronaghi, M ;

Pettersson, B ;

Nyrén, P .

PROTEIN EXPRESSION AND PURIFICATION, 1999, 15 (03) :381-388

[10] OPTICALLY DETECTED MAGNETIC-RESONANCE OF TRYPTOPHAN RESIDUES IN COMPLEXES FORMED BETWEEN A BACTERIAL SINGLE-STRANDED-DNA BINDING-PROTEIN AND HEAVY-ATOM MODIFIED POLY(URIDYLIC ACID) [J].

KHAMIS, MI ;

CASASFINET, JR ;

MAKI, AH ;

RUVOLO, PP ;

CHASE, JW .

BIOCHEMISTRY, 1987, 26 (12) :3347-3354

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