Difference between revisions of "20.109(S20):Identify clones to characterize (Day3)"
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==Introduction== | ==Introduction== | ||
| − | Today you will submit plasmids for sequencing analysis to determine the sequence differences between these clones and our characterized lysozyme binding scFv clone. These newly isolated plasmids encode for unique single-chain antibody fragment (scFv) clones that may show improved binding to our antigen, lysozyme | + | Today you will submit plasmids for sequencing analysis to determine the sequence differences between these clones and our characterized lysozyme binding scFv clone. These newly isolated plasmids encode for unique single-chain antibody fragment (scFv) clones that may show improved binding to our antigen, lysozyme. |
The invention of automated sequencing machines has made sequence determination a relatively fast and inexpensive process. 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 and 2000s. At the heart of sequencing reactions is chemistry worked out by Fred Sanger in the 1970s which uses dideoxynucleotides (image on left, below). 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” radioactive material was incorporated into the elongating DNA fragments so they could be visualized on X-ray film (image in center, below). 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 on right, below). Your sample was sequenced in this way by [http://www.genewiz.com/ Genewiz] on an ABI 3730x1 DNA Analyzer. | The invention of automated sequencing machines has made sequence determination a relatively fast and inexpensive process. 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 and 2000s. At the heart of sequencing reactions is chemistry worked out by Fred Sanger in the 1970s which uses dideoxynucleotides (image on left, below). 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” radioactive material was incorporated into the elongating DNA fragments so they could be visualized on X-ray film (image in center, below). 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 on right, below). Your sample was sequenced in this way by [http://www.genewiz.com/ Genewiz] on an ABI 3730x1 DNA Analyzer. | ||
Revision as of 17:28, 28 March 2020
Introduction
Today you will submit plasmids for sequencing analysis to determine the sequence differences between these clones and our characterized lysozyme binding scFv clone. These newly isolated plasmids encode for unique single-chain antibody fragment (scFv) clones that may show improved binding to our antigen, lysozyme.
The invention of automated sequencing machines has made sequence determination a relatively fast and inexpensive process. 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 and 2000s. At the heart of sequencing reactions is chemistry worked out by Fred Sanger in the 1970s which uses dideoxynucleotides (image on left, below). 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” radioactive material was incorporated into the elongating DNA fragments so they could be visualized on X-ray film (image in center, below). 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 on right, below). Your sample was sequenced in this way by Genewiz on an ABI 3730x1 DNA Analyzer.
Once we have the sequence of two new scFv clones, you will use SnapGene software to align the two new clone sequences to the original lysozyme binding scFv clone sequence. Once you know the difference(s) in the DNA sequence you can (1) determine where in the scFv the sequence difference occurs, (2) if the change in DNA sequence results in a difference in amino acid sequence and (3) predict how this difference in sequence affects lysozyme binding to the scFv clone.
Protocols
Reagents list
Next day: Characterize clone by titration using flow cytometry
