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| ==Introduction== | | ==Introduction== |
− | [[Image:Be109tracedata.jpg|right|thumb|160px|Sequence trace data]]
| + | dual plasmid system... |
− | [[Image:Be109dideoxynucleotide.jpg|thumb|left|450px|Normal bases versus chain-terminating bases]]
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− | [[Image:Be109sequencinggel.jpg|center|thumb|80px|Sequencing gel]]
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− | During this time, you will evaluate the DNA from your two X#Z candidates. The invention of automated sequencing machines has made sequence determination a relatively fast and inexpensive endeavor. The method for sequencing DNA is not new but automation of the process is recent, developed in conjunction with the massive genome sequencing efforts of the 1990s. At the heart of sequencing reactions is chemistry worked out by Fred Sanger in the 1970s which uses dideoxynucleotides (see schematic above left). These chain-terminating bases can be added to a growing chain of DNA but cannot be further extended. Performing four reactions, each with a different chain-terminating base, generates fragments of different lengths ending at G, A, T, or C. The fragments, once separated by size, reflect the DNA s sequence. In the old days (all of 15-20 years ago!) radioactive material was incorporated into the elongating DNA fragments so they could be visualized on X-ray film (image above center). More recently fluorescent dyes, one color linked to each dideoxy-base, have been used instead. The four colored fragments can be passed through capillaries to a computer that can read the output and trace the color intensities detected (image above right). Your sample was sequenced in this way by [http://www.genewiz.com/ Genewiz] on an ABI 3730x1 DNA Analyzer.
| + | aTc induction... |
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− | Analysis of sequence data is no small task. Sequence gazing can swallow hours of time with little or no results. There are also many web-based programs to decipher patterns. The nucleotide or its translated protein can be examined in this way. Thanks to the genome sequence information that is now available, a new verb, to BLAST, has been coined to describe the comparison of your own sequence to sequences from other organisms. BLAST is an acronym for Basic Local Alignment Search Tool, and can be accessed through [http://www.ncbi.nlm.nih.gov/ the National Center for Biotechnology Information (NCBI) home page].
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− | Today you will carefully examine the results of your sequencing reactions and determine which of your two samples contains the wanted mutation (and no unwanted random mutations).
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| ==Protocols== | | ==Protocols== |
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| #*The start and end of your sequencing may have several Ns. In this case it is best to omit these Ns by pasting only the 'good' sequence that is flanked by the ambiguous sequence. | | #*The start and end of your sequencing may have several Ns. In this case it is best to omit these Ns by pasting only the 'good' sequence that is flanked by the ambiguous sequence. |
| #Paste the pRSET-IPC or CaM sequence into the "Subject" box. | | #Paste the pRSET-IPC or CaM sequence into the "Subject" box. |
− | #Click on the BLAST button. Matches will be shown by vertical lines between the aligned sequences. You should see a long stream of matches, followed by lots of errors in the last ~200 bp of the sequence |