qPCR 引物篇(2)PCR 设计引物时如何查询基因

PCR 之引物设计--------基因序列查询篇 ?确定目标基因的形态：DNA or RNA ?查找目标基因

?比对目标基因同源性和特点 ?选取目标基因序列

?选取目标基因的目标区段

第一步：如何查基因：

1、mRNA 序列的查询； 以查大鼠cort为例；

http://www.ncbi.nlm.nih.gov/

search 选择 nucleotide for 1、rat cort 1、 在NCBI上搜索到目的基因，找到该基因的mRNA， Copy该ｍＲＮＡ序列作为软件查询序列的候选对

象。

该mRNA文件的命名： Rattusnorvegicuscortistatin (Cort), mRNA； NM_012835：是唯一的编号；

把以下序列存成ｔｘｔ文件：

Rattusnorvegicuscortistatin (Cort), mRNA

* Comment * Features * Sequence

LOCUS NM_012835 438 bp mRNA linear ROD 11-FEB-2008 DEFINITION Rattusnorvegicuscortistatin (Cort), mRNA. ACCESSION NM_012835

VERSION NM_012835.1 GI:6978682 KEYWORDS .

SOURCE Rattusnorvegicus (Norway rat) ORGANISM Rattusnorvegicus

Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Sciurognathi; Muroidea; Muridae; Murinae; Rattus. REFERENCE 1 (bases 1 to 438)

AUTHORS Xidakis,C., Mastrodimou,N., Notas,G., Renieri,E., Kolios,G., Kouroumalis,E. and Thermos,K.

TITLE RT-PCR and immunocytochemistry studies support the presence of somatostatin, cortistatin and somatostatin receptor subtypes in rat

Kupffer cells

JOURNAL Regul.Pept. 143 (1-3), 76-82 (2007) PUBMED 17481746

REMARK GeneRIF: RT-PCR and immunocytochemistry studies support the presence of cortistatin in rat Kupffer cells. REFERENCE 2 (bases 1 to 438)

AUTHORS Bourgin,P., Fabre,V., Huitron-Resendiz,S., Henriksen,S.J., Prospero-Garcia,O., Criado,J.R. and de Lecea,L.

TITLE Cortistatin promotes and negatively correlates with slow-wave sleep JOURNAL Eur. J. Neurosci. 26 (3), 729-738 (2007) PUBMED 17686045

REMARK GeneRIF: The capacity of CST-14 to increase SWA, together with preprocortistatin's inverse correlation with time spent in SWS, suggests a potential role in sleep homeostatic processes. REFERENCE 3 (bases 1 to 438)

AUTHORS Deghenghi,R., Avallone,R., Torsello,A., Muccioli,G., Ghigo,E. and Locatelli,V.

TITLE Growth hormone-inhibiting activity of cortistatin in the rat JOURNAL J. Endocrinol. Invest. 24 (11), RC31-RC33 (2001) PUBMED 11817718

REFERENCE 4 (bases 1 to 438)

AUTHORS de Lecea,L., Criado,J.R., Prospero-Garcia,O., Gautvik,K.M., Schweitzer,P., Danielson,P.E., Dunlop,C.L., Siggins,G.R., Henriksen,S.J. and Sutcliffe,J.G.

TITLE A cortical neuropeptide with neuronal depressant and sleep-modulating properties

JOURNAL Nature 381 (6579), 242-245 (1996) PUBMED 8622767

COMMENT PROVISIONAL REFSEQ: This record has not yet been subject to final NCBI review. The reference sequence was derived from U51919.1.

Summary: inhibits growth hormone secretion; may act as a neuropeptide to mediate signaling via a somatostatin receptor subtype [RGD].

FEATURES Location/Qualifiers source 1..438

/organism=\ /mol_type=\ /strain=\ /db_xref=\ /chromosome=\ /map=\

gene 1..438 /gene=\ /note=\ /db_xref=\ /db_xref=\ /db_xref=\ CDS 30..368 /gene=\

/note=\ /codon_start=1 /product=\ /protein_id=\ /db_xref=\ /db_xref=\ /db_xref=\ /db_xref=\

/translation=\ ATGGRRTGLLTFLAWWHEWASQDSSSTAFEGGTPELSKRQERPPLQQPPHRDKKPCKN

FFWKTFSSCK\ sig_peptide 30..110 /gene=\ mat_peptide 324..365 /gene=\

/product=\ORIGIN

1 aaagcacagacttcaggtctccaaggaggatgggtggctgcagcacaagaggcaagcggc 61 cgtcagccctcagtctgctgctgctgctgctgctctcggggatcgcagcctctgccctcc 121 ccctggagagcggtcccaccggccaggacagtgtgcaggatgccacaggcgggaggagga 181 ccggccttctgactttccttgcctggtggcatgagtgggcttcccaagacagctccagca 241 ccgctttcgaagggggtaccccggagctgtctaagcggcaggaaagaccacccctccagc 301 agcccccacaccgggataaaaagccctgcaagaacttcttctggaaaaccttctcctcgt 361 gcaagtagcccgagcctgaccggagcctgaccggccaccctgtgaatgcagccgtggcct 421 gaataaagagtgtcaagt //

其中origin后面的序列，是我们用来设计的序列。 要点：

1、 要会判断哪个是你要的序列；拉丁文

Mouse MUS。。。 Rat rattus。。。。

Human

2、 有别名，所以要判断是或者不是；

有受体，配体，名称中正好含这个关键词的； 3、如何判断： alsoknow as 4、多个转录本时如何查找同源序列；

还有其他内容，可以向BioTNT技术部垂询： 2、启动子序列的查询；

3、microRNA 序列的查询；

4、 SNP 序列的查询；