Obtaining sequence data is the first step in determining what the sequence of a

Question

Obtaining sequence data is the first step in determining what the sequence of a DNA strand is. It

is important to compare the reported, automated sequence with the sequence trace showing the peak

intensity at each nucleotide position. A first

step should always be to check the reported

Obtaining sequence data is the first step in determining what the sequence of a DNA strand is. It is important to compare the reported, automated sequence with the sequence trace showing the peak intensity at each nucleotide position. A first step should always be to check the reported AGOCTTTsequence against the appearance of the trace PS-18/ACS FOR O GGOCTONTTT C Question: Why? Question: What are options if you find discrepancies between the reported sequence and the appearance of the trace? There are a number of analyses that can be conducted on a DNA sequence. In this instance there are several things that we would like to know. First, did you clone the target sequence? Second, if the sequence is what was intended, how similar is it to homologs from other species at both the DNA and amino acid levels? Third, if you obtained multiple clones of the "same" target, how identical are they to each other?

Explanation / Answer

In sequencing reaction normal dNTPs and labeled dideoxy dNTPs are also present. During the sequencing reaction will terminate at various complimentary bases and that appear as form of signal. Generally post cleanup remove the non incorporated dNTPs and that will not interfere with sequence. A good intensity shows the sequencing has done and primers are working and good signal intensity also help to distinguish between signal to noise ratio. A higher difference between signals to noise ratio intensities increases the reliability of the data.

First thing is that the signals that comes during the sequencing from the complimentary bases hence you have to match it with the opposite strand or complimentary strand of the DNA.

Second possibility is that designed primers may bind to more than one place hence the more the one peak appears the same spot.

Incomplete post cleanup is important step for removal of extra labeled dNTPs they will also increase the signal to noise ratio.

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