A new reliable and sensitive nested PCR assay based on the human SRY gene for detection of interspecific chimeras

Jie YU; Kaijing LI; Main HUANG; Liping GUAN; Min ZHANG; Weihua LI; Jianfa HUANG; Ting LUO; Wencong WANG; Bikun XIAN; Xing LIU; Bing HUANG

Makale detayları

A new reliable and sensitive nested PCR assay based on the human SRY gene for detection of interspecific chimeras

Yazarlar:

Jie YU | Kaijing LI | Main HUANG | Liping GUAN | Min ZHANG | Weihua LI | Jianfa HUANG | Ting LUO | Wencong WANG | Bikun XIAN | Xing LIU | Bing HUANG

Yayıncı:

TUBITAK

Dergi:

Turkish Journal of Biology 2016 Vol.40 Issue 4, pp.736-746

Belge Tipi:

Makale

Yayın Tarihi:

7/2016

Belge Dili:

Türkçe

Document ID (Org No.):

2000811599

Sıklık:

İki Aylık

eISSN:

1303-6092

pISSN:

1300-0152

Özet:

 

Although interspecific chimeras can be identified using laborious techniques, more accurate and rapid detectable methods need to be established. The present study develops a nested polymerase chain reaction (nPCR) assay to detect human-mouse chimeras. A set of previously validated outer primers, specific to the human SRY gene, was used for conventional one-step PCR, while the inner primers for nPCR were designed. The specificities of PCR and nPCR assays were examined; both primer sets yielded PCR amplification products from male human epidermis-derived mesenchymal stem cell-like pluripotent cell DNA but no amplification products from negative control DNA. The sensitivity of this nPCR was determined using mixed DNA. Measurable amplification of SRY transcripts was a male human to female mouse DNA ratio of 1:10,000. We then tested the nPCR assay on tissues from female mouse chimeras. The nPCR products were selected randomly for sequencing and positive samples were further analyzed by fluorescence in situ hybridization (FISH) using specific probes for the human SRY gene and by immunofluorescence staining for species-specific markers. There was 100% concordance among nPCR, FISH, and immunofluorescence results, and the nPCR product sequences were consistent with the human SRY gene. Taken together, we developed a new, highly specific, sensitive, and reliable method to detect human-mouse chimeras.

Makale detayları

A new reliable and sensitive nested PCR assay based on the human SRY gene for detection of interspecific chimeras

Yazarlar: Jie YU | Kaijing LI | Main HUANG | Liping GUAN | Min ZHANG | Weihua LI | Jianfa HUANG | Ting LUO | Wencong WANG | Bikun XIAN | Xing LIU | Bing HUANG

Yayıncı: TUBITAK

Dergi: Turkish Journal of Biology 2016 Vol.40 Issue 4, pp.736-746

Belge Tipi: Makale

Yayın Tarihi: 7/2016

Belge Dili: Türkçe

Document ID (Org No.): 2000811599

Sıklık: İki Aylık

eISSN: 1303-6092

pISSN: 1300-0152

Sayı

Yazarlar:

Jie YU | Kaijing LI | Main HUANG | Liping GUAN | Min ZHANG | Weihua LI | Jianfa HUANG | Ting LUO | Wencong WANG | Bikun XIAN | Xing LIU | Bing HUANG

Yayıncı:

TUBITAK

Dergi:

Turkish Journal of Biology 2016 Vol.40 Issue 4, pp.736-746

Belge Tipi:

Makale

Yayın Tarihi:

7/2016

Belge Dili:

Türkçe

Document ID (Org No.):

2000811599

Sıklık:

İki Aylık

eISSN:

1303-6092

pISSN:

1300-0152