Sequencing through homopolymer regions

Timothy A. Rawlings rawlings at fiu.edu
Tue Nov 18 11:03:55 EST 1997


Greetings
	Recently I have been experiencing problems sequencing through relatively
short homopolymer regions (10 - 12 bases of As or Ts) of PCR amplified
genomic DNA using dye terminators and Amplitaq-FS chemistries on our ABI
377.  The sequence on both strands becomes unreadable immediately following
this homopolymer stretch.  Our local Applied Biosystems representative has
suggested that this difficulty might result from enzyme slippage during PCR
as opposed to during cycle sequencing.  I have noticed similar problems
reported on the Newsgroup during the past year or so , but have not  read
any messages indicating the specific cause of this problem or methods that
have successfully resolved this difficulty without the use of polyA/poly T
primers or resorting to manual sequencing!   
	I would very much appreciate hearing from anyone who has successfully
managed to trace this problem and to find ways (through PCR conditions,
sequencing chemistries, etc.) to successfully sequence through these
regions. 	

	Cheers, Tim Rawlings


Timothy A. Rawlings
Department of Biological Sciences,
OE 246,
Florida International University,
University Park,
Miami, FL 33199, USA
Tel: (305) 348-3110 (Lab); (305) 774-1378 (Home)
FAX: (305) 348-1986
E-mail: rawlings at fiu.edu
	




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