<html><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; "><div>Hi there,</div><div>I am totally unsurprised that you are finding sequence differences with the gene you cloned and what is in NCBI. We see this in the genes we clone in the lab - one to 10 nucleotide sequence differences (our genes are 1500 bp) with aa changes. You can see this if you pull out sequence information from NCBI for which there are multiple entries - they will not be completely identical. Some of this is strain differences. It doesn't mean it won't be expressed. You need to be sure that the differences are real (sequence several clones so that you can show it isn't a PCR or sequencing error) and that it will translate in frame without stops. </div><div>Joanna Wilson</div><div><br></div><div>Re:</div>Message: 1<br>Date: Fri, 18 Apr 2008 11:34:55 -0700 (PDT)<br>From: <a href="mailto:dykvar@yahoo.com">dykvar@yahoo.com</a><br>Subject: [Zbrafish] Sequence homology for protein expression - help!<br>To: <a href="mailto:bionet-organisms-zebrafish@moderators.isc.org">bionet-organisms-zebrafish@moderators.isc.org</a><br>Message-ID:<br><span class="Apple-tab-span" style="white-space: pre; "> </span><<a href="mailto:9da02037-1cd7-4aee-a587-463a246279c4@e39g2000hsf.googlegroups.com">9da02037-1cd7-4aee-a587-463a246279c4@e39g2000hsf.googlegroups.com</a>><br>Content-Type: text/plain; charset=windows-1252<br><br>Hi,<br>I have a general question regarding rescue and protein expression via<br>construct in ZF. To do this one must first clone the protein mRNA<br>sequence from cDNA, then insert into an expression vector (I’m using<br>PCS2+). However, whenever I clone and then sequence I never get 100%<br>homology when blasting on NCBI. Although I use high-fidelity (Roche)<br>enzyme, I get about 4-5 nucleotides different (out of ~900) and 2-3 aa<br>different from what is in the database.<br>Is this normal? Is it a result of polymorphism? Or is it a problem<br>with my methodology? And what degree of homology is needed (assuming<br>no gaps exist) for the protein being expressed properly?<br>Your help is appreciated!!<br><br><br><div apple-content-edited="true"> <span class="Apple-style-span" style="border-collapse: separate; border-spacing: 0px 0px; color: rgb(0, 0, 0); font-family: Helvetica; font-size: 20px; 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 style="word-wrap: break-word; -khtml-nbsp-mode: space; -khtml-line-break: after-white-space; "><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 style="font-size: 12px; "><span class="Apple-style-span" style="font-size: 12px; ">Dr. Joanna Wilson</span></div><div style="font-size: 12px; "><span class="Apple-style-span" style="font-size: 12px; ">Assistant Professor</span></div><div style="font-size: 12px; "><span class="Apple-style-span" style="font-size: 12px; ">Department of Biology</span></div><div style="font-size: 12px; "><span class="Apple-style-span" style="font-size: 12px; ">McMaster University</span></div><div style="font-size: 12px; "><br class="khtml-block-placeholder"></div><div style="font-size: 12px; "><span class="Apple-style-span" style="font-size: 12px; ">1280 Main Street West</span></div><div style="font-size: 12px; "><span class="Apple-style-span" style="font-size: 12px; ">Hamilton ON</span></div><div style="font-size: 12px; "><span class="Apple-style-span" style="font-size: 12px; ">L8S 4K1</span></div><div style="font-size: 12px; "><br class="khtml-block-placeholder"></div><div style="font-size: 12px; "><span class="Apple-style-span" style="font-size: 12px; ">Tel: 905-525-9140 ext 20075</span></div><div style="font-size: 12px; "><span class="Apple-style-span" style="font-size: 12px; ">Fax: 905-522-6066</span></div><div style="font-size: 12px; "><span class="Apple-style-span" style="font-size: 12px; "><a href="mailto:joanna.wilson@mcmaster.ca">joanna.wilson@mcmaster.ca</a></span></div><div style="font-size: 12px; "><br class="khtml-block-placeholder"></div><br class="Apple-interchange-newline"></span></span></div></span> </div><br></body></html>
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