I am forwarding this question from a user of our genotyping facility at Wake Forest. Please feel free to reply directly to Dr. Yu or to the bulletin board. I will forward all replies to him that are posted. Thanks in advance for your help.
Mark Lively
>>>>>Question:
I am writing you about the 'bleed-through' problem we are experiencing in our automated genotyping effort. Our genotyping is being carried out on an ABI 377 sequencer. We are using the CHLC/Weber genome screening set purchased from Research Genetics. The forward primers are labeled with three dyes: FAM for blue, TET for green and HEX for yellow. The problem is that the blue marker peaks are also present as green peaks, resulting in more peaks in the electropherogram. There is also a lot of noise in the background when the green peaks are viewed.
Because of these problems, it is difficult to distinguish in data analysis between the true peaks derived from the green markers and those contaminating peaks from the blue markers. We have gotten contradictory advice ranging from using fewer PCR products when loading green markers to using the new dye NED in place of TET. We have found that a reliable but tedious method to solve the problem is to generate a new matrix regularly. We are wondering whether there are other people who are experiencing the same
problem and whether there is a better way to solve it.
Sincerely,
Hongrun Yu, PhD
Dept of Biochemistry
Wake Forest Univ Sch of Med
Winston-Salem NC 27157
Phone: (336) 716-9057
E-mail: yu@invader.bgsm.wfu.edu