In Situ Cycle Runoffs
14 October 1994 ECK
Revised 29 November 1995 ECK
Message runoffs locate the 5' terminus of mRNA. The standard technique is simple. A labelled DNA primer, complementary to a region near the 5' end of the message, is annealed to whole-cell RNA and extended by reverse transcriptase. Products are run ajacent to a sequence ladder made with the same primer. The terminus is found by referencing extension bands to the sequence. The technique is not a common one because of the difficulty of preparing message undegraded by endogenous nucleases and other activities, such as ubiquitous "finger-tip" endonuclease. Interpretation of results can also be complicated by template secondary structure, which impedes the progress of the enzyme.
The technique of in situ cycle runoffs uses intact living bacteria as template source. It is useful only for nonthermophilic organisms, such as Escherichia coli or Salmonella typhimurium. Cells are suspended in primer extension mix and lysed by a brief exposure to high temperature. This denatures endogenous or contaminating nucleases. During cycling, temperatures are kept high enough to maintain this inhibition. This also disrupts most intrinsic template secondary structure. Temperature-stable Tth DNA polymerase/reverse transcriptase is used to extend hybridized primers.
Reactions are packaged in glass capillaries and cycled in an Air ThermoCycler (Idaho Technologies, PO Box 50819, Idaho Falls, ID 83402). The choice of machine may be important as it ramps quickly between temperatures. Dwell times at denaturation and annealing temperatures are zero. This allows rapid cycling with increased stringency. In addition, overall time spent near denaturation temperature is low, minimizing enzyme aging and spontaneous template hydrolysis. We have not tested the protocal on other machines.
Oligonucleotides should follow the normal rules for good PCR primers, with melting temperatures in the range 70-80 deg.
Technique developed by Eric Kofoid & Jeff Lawrence, October 1994.
1. Bacterial cells
Dilute an overnight culture 1:100 and grow to Klett 70.
Chill on ice.
Pellet cells gently, resuspend in 1 vol ddH2O.
Pellet again and resuspend in 5 vol ddH2O.
2. Primer end-labelling
8 microL ddH2O
4 microL 5 microM primer
2 microL 10x Kinase Buffer (NEB)
1 microL 10 u/microL T4 DNA Kinase (NEB)
5 microL [gamma32P]ATP
15' x 37 deg
5' x 70 deg (to kill enzyme)
1 microL 20 mg/mL glycogen (or other carrier)
2 microL 7.5 M NH4OAc
58 microL 95% ethanol
10' x ice
Pellet 10' in microcentrifuge.
Wash once with 70% ethanol.
Lightly vacuum off ethanol without drying pellet.
Resuspend in 20 microL ddH2O.
Can be stored at -20 deg.
3. Primer extension
9.5 microL ddH2O
2 microL washed cells
2 microL 10x Reverse Transcriptase Buffer (Promega)
2 microL 10 mM MnCl2
2 microL 2 mM 4 dNTP's
0.5 microL Tth DNA polymerase/RT transcriptase (Promega)
2 microL [32P] labelled primer
Seal reaction in plastic capillaries, available from Idaho Technologies.
(Used to minimize potential contamination.)
Typical thermocycler settings:
30" x 94 deg
25 - 100 cycles: 0" x 94 deg, 0" x 55 deg, 2' x 72 deg
5' x 72 deg
(Cycles, annealing temp. & extension time can be varied as needed.)
Dry down under vacuum.
Can be stored at -20 deg.
Run extension reaction side by side with dideoxy sequences of
corresponding DNA. Band position in the sequence determines mRNA 5' end.
Pharmacia, "Ultrapure dNTP Set", cat. 272035
Prepare mix as follows:
92 microL H2O
2 microL each of dATP, dCTP, dGTP & dTTP
0.3% xylene cyanol
0.3% bromophenol blue
100 mM Na2EDTA @ pH 7.0 in formamide.
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eckofoid at ucdavis.edu