A cDNA-AFLP protocol with reciprocally arranged 2-enzyme sequential digestion and silver staining detection

Mehmet Ali SÜDÜPAK

Makale detayları

A cDNA-AFLP protocol with reciprocally arranged 2-enzyme sequential digestion and silver staining detection

Yazarlar:

Mehmet Ali SÜDÜPAK

Yayıncı:

TUBITAK

Dergi:

Turkish Journal of Biology 2014 Vol.38 Issue 2, pp.260-270

Belge Tipi:

Makale

Yayın Tarihi:

5/2014

Belge Dili:

Türkçe

Document ID (Org No.):

2000812261

Sıklık:

İki Aylık

eISSN:

1303-6092

pISSN:

1300-0152

Anahtar kelimeler:

Özet:

 

This study provides a modified protocol to the standard cDNA-amplified fragment length polymorphism (AFLP) procedure that has been used to detect differentially expressed transcripts in various systems. Modifications aimed to improve the overall coverage of the technique and the isolation of single fragments from each of the cDNA species. The protocol utilizes oligo-d(T) coupled magnetic beads to isolate and synthesize, and 2-enzyme sequential digestions of ds-cDNA molecules with flip-flop strategy on the beads to release transcript tags representing individual mRNA type and quantity. The protocol has been applied to detect Puccinia sorghi-induced expressional changes in maize leaf material sampled at 8 time points following inoculation. cDNA-AFLP analysis of this material pooled into 2 time intervals (6-24 h and 36-96 h), along with their controls, revealed banding patterns in which 10-20 differentially expressed message tags were present among the 40-80 bands detected per primer combination. Three hundred and ten differentially expressed message tags were sequenced, and the majority with known functions were found to be associated with plant-microbe interactions. Although it requires a few additional steps, this protocol appears to be effective in revealing differentially expressed messages, and the study demonstrates that pooling the material in the preparation of templates and silver staining detection provides economical alternatives for surveying and identifying expressional modulations occurring over relatively longer time periods.

 

This study provides a modified protocol to the standard cDNA-amplified fragment length polymorphism (AFLP) procedure that has been used to detect differentially expressed transcripts in various systems. Modifications aimed to improve the overall coverage of the technique and the isolation of single fragments from each of the cDNA species. The protocol utilizes oligo-d(T) coupled magnetic beads to isolate and synthesize, and 2-enzyme sequential digestions of ds-cDNA molecules with flip-flop strategy on the beads to release transcript tags representing individual mRNA type and quantity. The protocol has been applied to detect Puccinia sorghi-induced expressional changes in maize leaf material sampled at 8 time points following inoculation. cDNA-AFLP analysis of this material pooled into 2 time intervals (6-24 h and 36-96 h), along with their controls, revealed banding patterns in which 10-20 differentially expressed message tags were present among the 40-80 bands detected per primer combination. Three hundred and ten differentially expressed message tags were sequenced, and the majority with known functions were found to be associated with plant-microbe interactions. Although it requires a few additional steps, this protocol appears to be effective in revealing differentially expressed messages, and the study demonstrates that pooling the material in the preparation of templates and silver staining detection provides economical alternatives for surveying and identifying expressional modulations occurring over relatively longer time periods.

Makale detayları

A cDNA-AFLP protocol with reciprocally arranged 2-enzyme sequential digestion and silver staining detection

Yazarlar:

Mehmet Ali SÜDÜPAK

Yayıncı:

TUBITAK

Dergi:

Turkish Journal of Biology 2014 Vol.38 Issue 2, pp.260-270

Belge Tipi:

Makale

Yayın Tarihi:

5/2014

Belge Dili:

Türkçe

Document ID (Org No.):

2000812261

Sıklık:

İki Aylık

eISSN:

1303-6092

pISSN:

1300-0152

Key words: Maize

P. sorghi

plant-microbe interaction

cDNA-AFLP

differential gene expression

Sayı

Makale detayları

A cDNA-AFLP protocol with reciprocally arranged 2-enzyme sequential digestion and silver staining detection

Yazarlar: Mehmet Ali SÜDÜPAK

Yayıncı: TUBITAK

Dergi: Turkish Journal of Biology 2014 Vol.38 Issue 2, pp.260-270

Belge Tipi: Makale

Yayın Tarihi: 5/2014

Belge Dili: Türkçe

Document ID (Org No.): 2000812261

Sıklık: İki Aylık

eISSN: 1303-6092

pISSN: 1300-0152

Key words: Maize

P. sorghi

plant-microbe interaction

cDNA-AFLP

differential gene expression

Sayı

Yazarlar:

Mehmet Ali SÜDÜPAK

Yayıncı:

TUBITAK

Dergi:

Turkish Journal of Biology 2014 Vol.38 Issue 2, pp.260-270

Belge Tipi:

Makale

Yayın Tarihi:

5/2014

Belge Dili:

Türkçe

Document ID (Org No.):

2000812261

Sıklık:

İki Aylık

eISSN:

1303-6092

pISSN:

1300-0152