New primer combinations with comparable melting temperatures detecting highest numbers of nosZ sequences from sequence databases
Stres, Blaž (Author), Murovec, Boštjan (Author)

URLURL - Presentation file, Visit http://aas.bf.uni-lj.si/zootehnika/94-2009/PDF/94-2009-2-139-142.pdf This link opens in a new window

We explored existing primer sequences targeting nitrous oxide reductase (nosZ) gene in order to explore their capability to recognize variant nosZ sequences. Published nosZ sequences longer than 380 AA residues were obtained from FunctionalGene Database /Repository (http://flyingcloud.cme.msu.edu/fungene/) and used for explorations with PrimerChart program. The numbers of sequences recovered using all possible forward and reverse primer combinations were determined and the stringency of primer site recognition was further varied by allowing 1, 2, or 3 primer mismatches to DNA binding site. We identified novel primer combinations resulting in satisfactory amplicon length (> 500 bp) and increased sequence recognition capabilities at comparable forward and reverse primer melting temperatures. Overall, this study indicates that current state of the art molecular methods can be and should frequently be further refined by the use of targeted bioinformatic approaches.

Keywords:micorbiology, molecular biology, denitrification, nitrous oxide reductase, melting temperature, detection
Work type:Not categorized (r6)
Tipology:1.01 - Original Scientific Article
Organization:BF - Biotechnical Faculty
Number of pages:str. 139-142
Numbering:Letn. 94, št. 2
ISSN on article:1581-9175
COBISS.SI-ID:2546568 Link is opened in a new window
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Record is a part of a journal

Title:Acta agriculturae Slovenica
Shortened title:Acta agric. Slov.
Publisher:Biotehniška fakulteta
COBISS.SI-ID:213840640 This link opens in a new window

Secondary language

V tej študiji sva raziskala obstoječe sekvence začetnih oligonukleotidov, s katerimi se pomnožujejo fragmenti gena za reduktazo N2O (nosZ), da bi proučila njihovo zmožnost prepoznavanja variant sekvenc nosZ. Objavljene sekvence gena nosZ daljše od 380 aminokislninskih ostankov sva pridobila od FunctionalGene Database /Repository (http://flyingcloud.cme.msu.edu/fungene/) in jih analizirala s programom PrimerChart. Raziskala sva število, ki ga prepoznajo posamične mone kombinacije yačetnih oligonukleotidov. V nadaljevanju sva spreminjala natančnost prileganja začetnih oligonukleotidov na tarčno DNK tako, da sva dovolila 1, 2, or 3 napačna parjenja med začetnim oligonukleotidom in DNK. Tako sva identificirala nove kombinacije začetnih oligonukleotidov, ki ustvarijo ustrezno dolge fragmente (> 500 bp), s povišano sposobnostjo prepoznavanja sekvenc pri primerljivi temperaturi taljenja začetnih oligonukleotidov. Prav tako so se nakazale nove možnosti za izboljšanje začetnih oligonukleotidov z vnosom novih degeneriranih mest. Ta študija nakazuje, da je novejše molekularne metode možno in tudi potrebno pogosto nadgrajevati s ciljanimi bioinformatskimi pristopi.

Keywords:mikrobiologija, molekularna biologija, denitrifikacija, dušikov oksid, reduktaza, temperatura taljenja, zaznavanje

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