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<div>Re: Sequence homology for protein expression</div><div><br></div>Dear E.A.,<div><br></div><div>I would say that to be careful, you should consider different amino acid substitutions in different clones to be a problem. For the following reasons:</div><div><br></div><div>1) The fact that you are not getting the same errors clone-to-clone strongly suggests that you are getting PCR errors. High fidelity enzymes are not perfect. Are you running high numbers of amplification cycles (>30-32)? The less cycles, the less errors you should get, although you can still be unlucky and get errors at low numbers of cycles. You could try: using a variety of enzymes, I now prefer Phusion (from Finnzymes) and iProof (from Bio-Rad), they seem to give me the lowest number of errors. Making all your cDNA from a single clutch from a pairwise cross. Then you will know that you should get very few polymorphisms clone-to-clone.</div><div><br></div><div>2) Many amino acid substitutions don't matter, but without having the "wild-type" to compare functionality against, it will be hard to be sure yours doesn't. Especially a Threonine to Alanine substitution, since Thr is frequently a phosphorylated amino acid for protein regulation. If I start getting "polymorphisms" different from the expected amino acid sequence, I sequence quite a few more clones to make sure they all have the same substitutions. I don't worry so much about silent base substitutions (in the third position of the codon leading to no change in amino acid).</div><div><br></div><div>In general, I don't think pairwise BLAST is a very good way to assess whether your clones are good. The BLAST algorithm tends to cut off ends or regions that are "not conserved" so you don't get a base-by-base comparison. We use the Sequencher software here. I'm not sure if you have access to it, but many universities and companies have purchased networked licenses that you can use on a client/server basis using downloaded software. I think it's the best thing going. You can make a "virtual" clone of your expected sequence (in the vector) and then do an alignment (make a contig) against the actual sequencing returned. Then you can look base-by-base at which bases match and which do not. In addition, you can consider the quality of the sequencing data. If your "polymorphisms" are towards the end, or very beginning, of a sequencing run, the sequence quality may be pretty bad. If so, you're clone could be correct and you're getting fooled by bad sequence. You can't assess this parameter doing a pairwise BLAST. If you don't have Sequencher, I would still recommend trying to find some sort of sequence analysis software, there's quite a bit out there for free.</div><div><br></div><div>Good luck.</div><div><br></div><div>Wilson</div><div><div><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; text-align: auto; -khtml-text-decorations-in-effect: none; text-indent: 0px; -apple-text-size-adjust: auto; text-transform: none; orphans: 2; white-space: normal; widows: 2; word-spacing: 0px; "><div>--------------------------------------------------------------------------------------------</div><div>Wilson Clements, Ph.D. </div><div><br class="khtml-block-placeholder"></div><div><a href="mailto:wclements@ucsd.edu">wclements@ucsd.edu</a></div><div><br class="khtml-block-placeholder"></div><div>Dept. of Biology </div><div>Section of Cell and Developmental Biology </div><div>University of California at San Diego </div><div>9500 Gilman Dr.</div><div>Natural Sciences Building 6105</div><div>La Jolla, CA 92093-0380</div><div><br class="khtml-block-placeholder"></div><div>TEL (858) 534-6955</div>LAB (858) 822-4658<br class="Apple-interchange-newline"></span></span><div>FAX (858) 822-5740</div><br class="Apple-interchange-newline"></span></span></span></span></span></span> </div><div><br><div>Begin forwarded message:</div><br class="Apple-interchange-newline"><blockquote type="cite"><p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica">Hi Joanna,</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px; font: 12.0px Helvetica; min-height: 14.0px"><br></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica">Thanks for the answer. The strange thing is that I do not get the same</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica">‘errors’ when sequencing multiple clones. I can only think that this</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica">is due to the fact that the cDNA was made from multiple larvae</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica">? Or</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica">that although I used HF the enzyme is causing errors. In any case,</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica">when I use blastx for protein sequence (part of the protein – 900bp) I</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica">get something like this:</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica"><span class="Apple-converted-space"> </span>Score =<span class="Apple-converted-space"> </span>446 bits (1147),<span class="Apple-converted-space"> </span>Expect = 1e-123</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica"><span class="Apple-converted-space"> </span>Identities = 220/221 (99%), Positives = 220/221 (99%), Gaps = 0/221</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica">(0%)</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica"><span class="Apple-converted-space"> </span>Frame = +2</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px; font: 12.0px Helvetica; min-height: 14.0px"><br></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica">(One alanine substituting a threonine)</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica">Is such a substitution a problem?</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica">Thanks very much</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px"><font face="Helvetica" size="3" style="font: 12.0px Helvetica">E.A.</font></p> <p style="margin: 0.0px 0.0px 0.0px 0.0px; font: 12.0px Helvetica; min-height: 14.0px"><br></p> </blockquote></div><br></div></body></html>
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