An article for ABI310 users

David David
Tue Jul 1 08:22:34 EST 1997


Compatibility of different extension product purification protocols =
with
sequencing analysis using ABI 310 Genetic Analyzer

Aim: To evaluate the suitability of various extension product
purification protocols for use with ABI 310 which is capillary-based.

Materials and Method
ABI PRISM Dye Terminator Kit with AmpliTaq FS, pGEM-3Zf(+), M13(-21)
primer, 70% ethanol, sodium acetate-containing 95% ethanol,
MgCl2-containing 70% ethanol, Centri-Sep columns, GeneAmp PCR 2400
cycler, ABI Template Suppression Reagent (TSR), ABI POP-6 polymer,
molecular biology grade calf intestinal alkaline phosphatase and 10X
buffer (New England BioLabs). 
  20ul aliquots were dispensed from a master mix into six microtubes.
Each aliquot contained 8ul of Ready-Reaction Mix, 400ng pGEM-3Zf(+), =
and
3.2pmol M13(-21) primer. Sequencing reactions were performed using
GeneAmp PCR 2400 cycler as described in the manufacturer=92s =
protocol[1].
To account for any between-sample variations, the reacted samples were
pooled and then aliquoted in 20ul each into six fresh tubes. For each
tube, a different protocol was used to purify the extension products.
Protocols used were outlined as follows:
(A)Centri-Sep spin column purification[1]
(B)Sodium acetate containing ethanol precipitation[1]
(C)95% ethanol precipitation[2]
(D)MgCl2 containing ethanol precipitation[3]
(E)Alkaline phosphatase digestion[4] plus method C
(F)Method D followed by method A

The dried extension products in each tube were reconstituted with 24ul
TSR and  heated at 95C for 2min before loading onto the autosampler in
the ABI310. The instrument was equipped with a POP-6 polymer-filled
capillary (47cm X 50um). DNA was electrokinetically injected into the
capillary at 2.0kV for 30sec and separated at 15kV for 30min at 50oC.
All sequencing traces were analyzed with ABI PRISM Sequencing Analysis
Software v2.0.2.

Results
	Results		
Methods	Signal quality	Read length (bp)	Average Signal Intensity 
A	highly readable		429		G:318 A:477 T:297 C:178

B	signal strength 	400		G:144 A:239 T:149 C:88
	declined after 
	the first 300
	bases		

C	poor; frequent		300		G:337 A:583 T:537 C:450
	interference 
	from dye-blobs	

D	poor;unreadable		0		G:31 A:42 T:102 C:33

E	no signal		0		not available

F	poor; high 		100		comparable to method D
	background		

Conclusion
  Purification of extension products using either Centri-Sep spin
columns or  sodium acetate-containing ethanol precipitation remained =
to
be the two most recommended methods to use with ABI310. Although =
method
D which involved the use of MgCl2 has been used successfully with
ABI377, it gave no signals when used with ABI310. It is possible that
the residual salt interfered with the electrokinetic injecting =
mechanism
in ABI310. However, it is still puzzling as to why method E gave no
signals at all since the 95% ethanol step should wash off any residual
salt (mainly TrisHCl) that came with the phosphatase buffer. Maybe =
Tris
salt is less hydrophilic when mixed with 95% ethanol making it more
difficult to be completely eliminated from the DNA pellet. 

References
1. ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit =
Protocol
(P/N 402078) Revision A. August 1995.
2. ABI PRISM Dye Primer Cycle Sequencing Ready Reaction Kit Protocol.
July 1995.
3. ABI PRISM Dye Rhodamine Sequencing Protocol. 1997.
One unit of alkaline phosphatase and 2ul of 10X enzyme buffer was =
added
to each 20ul reaction. Mix well by pipetting and incubate at 37oC for
30min.

Acknowledgement 
I would like to thank Kelly Gallagher, Mark Holloway, Richard Harrison
et al. from Perkin-Elmer Australia Technical Support Group for their
technical advice.

David CY Fung
Department of Molecular Genetics
The Royal Prince Alfred Hospital
Camperdown NSW 2050
Australia



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