John M. S. Bartlett.pdf - Bio-Nica.info

Nested RT-PCR in a Single Closed Tube 155
Table 1
Volume and Concentration of Reactives for CTV Detection by Nested RT-PCR
Ingredients Cocktail A (RT-PCR) Cocktail B (nested-PCR)
10X RT-PCR buffer to 13.00 µl to 11.00 µl
MgCl 2 (25 mM) to 13.60 µl –
dNTPs (5mM each) to 12.25 µl –
Triton ® X-100 (4%) to 12.50 µl –
3′ external primer (100 µM) to 10.15 µl 3′ internal primer (100 µM) to 10.80 µl
(Pe1 in Fig. 1) (Pi1 in Fig. 1)
5′ external primer (100 µM) to 10.15 µl 5′ internal primer (100 µM) to 10.80 µl
(Pe2 in Fig. 1) (Pi2 in Fig. 1)
DMSO (100%) to 11.50 µl –
H 2 O to 30.00 µl to 10.00 µl
AMV (10 U/µl) to 10.20 µl –
Taq Pol (5 U/µl) to 10.20 µl –
3. Methods
3.1. Primer Design (see Note 5)
Sequenced regions of each RNA target can be recovered using the Nucleotide
Sequence Search program located in the Entrez Browser program provided by the
National Center for Biotechnology Information (NCBI) (http://www3.ncbi.nlm.nih.gov/
Entrez; Bethesda, MD). Conserved regions for each target can be studied using the
similarity search Advanced BLAST 2.0, with the blast program designed to support
analysis of nucleotides (http://www3.ncbi.nlm.nih.gov/blast/blast.cgi?Jform=1) (25).
The alignment view could be performed as master-slave with identities to analyze
significant nucleotide homologies in the molecular data retrieved from NCBI’s
integrated databases, GenBank, EMBL, and DDBJ. Specific nucleotide regions should
be selected. Different specific primers with similar annealing temperature can be
subsequently designed for the target of interest, based on Oligo software utility
(http://www.lifescience-software.com/oligo.htm; LRS, Long Lake, MN). By using
this methodology, appropriate PCR primers to different RNA targets can be easily
and properly designed.
3.2. Nested PCR Product Generation
1. RT-PCR and nested PCR cocktail preparation (see Fig. 2).
a. When the annealing temperatures between external and internal primers are very
different (external 45°C and internal 60°C) and the external primers do not anneal at
60°C (for example, CTV detection), see Table 1.
b. When the annealing temperatures of the external and internal primers are similar
(external 50°C and internal 60°C) and the external primers anneal at 60°C (for example,
PPV detection), see Table 2.
2. Add 30 µL of cocktail A for reverse transcription and external amplification to the bottom
of an 0.5-mL Eppendorf tube.
3. Introduce the small plastic cone (see Fig. 2 and Notes 1 and 4) and add 10 µL of cocktail
B into the cone.