PCR Reaction in assessing mRNA expression

Francis Kuttner dirt_spot at yahoo.com
Mon Apr 3 06:06:37 EST 2000


Hi, I have a question about PCR reaction to assess mRNA expression
(semiquantitative) of this particular gene.

Potential Primers:
A. 5'-GTCATGTGTGTGGAGAGCGT-3'
B. 5'-GACAACATCGCCCTGTGGAT-3'
C. 5'-GCAGGCATGTTGACTTCACT-3'
D. 5'-GGCAGGCATGTTGACTTCAC-3'

Primer Parameters:
primer name              A          B           C            D
location                210        403         533          780
length                20 nuc.     20 nuc.     20 nuc.      20 nuc.
%GC                     55%        55%          50%          55%
Tm (1/2 melting temp)   69C        73C          69C          71C
runs of GC at 3' enc     0           0           0            1
runs of any bases        2           3           2            2
hairpins                 0           0           0            0


Primer interactions:
(length in nucleotides)   3' end involved         any interaction

                              A  B  C  D              A  B  C  D
                          A   1  2  2  3          A   4  4  4  4
                          B      3  2  3          B      3  6  6
                          C         2  2          C         4  4
                          D            2          D            4


Partial Gene Map with Intron/Exon Structure and Primer Locations:

   A --->           B --->
------------       ---------------------------
------------
| exon 2   |-------|         exon 3          |----------------------|
exon 4  |
------------       ---------------------------
------------
                                        <--- C
<--- D


I believe that A and C, B and D primers go together yet I am confused
about whether to use the primer dimers that involves 3' ends or any type
of primer dimers.  If anyone who is familiar with these research
techniques can elucidate this problem including cDNA primer, PCR
primers, primer concentration, MgCl2 conc., controls, and thermal cycle
parameters (temp, time, # cycles) your help is appreciated.  Your
comments and suggestions are welcome, please if you have any information
or can refer me to a text manual or some literature please feel free to
email me at dirt_spot at yahoo.com.

Thanks,
Francis
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