Southern blot: Difference between revisions
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The DNA to be tested must be cut into pieces by a [[restriction enzyme]] that cleaves the DNA at specific sites in the DNA sequence. To sort the complex mixture of DNA fragments by size the mixture of DNA is passed through an [[agarose]] gel using an electrical current. The process is called [[gel electrophoresis]]; small pieces of DNA move rapidly through the gel than larger pieces. | The DNA to be tested must be cut into pieces by a [[restriction enzyme]] that cleaves the DNA at specific sites in the DNA sequence. To sort the complex mixture of DNA fragments by size the mixture of DNA is passed through an [[agarose]] gel using an electrical current. The process is called [[gel electrophoresis]]; small pieces of DNA move rapidly through the gel than larger pieces. | ||
The DNA trapped in the gel is treated with [[alkali]] to denature the DNA fragments to single stranded pieces. Then, by wicking liquid through the gel (blotting), the DNA is | The DNA trapped in the gel is treated with [[alkali]] to denature the DNA fragments to single stranded pieces. Then, by wicking liquid through the gel (blotting), the DNA is transferred to a [[nitrocellulose filter]] or charged [[nylon membrane]]. The DNA is attached to the filter permanently by either heating or crosslinking with UV light. Once attached to the filter the relative location of the DNA fragments from the agarose gel are preserved on the filter. | ||
To identify the location (size) of a specific piece of DNA on the filter a sequence specific probe needs to be made. The probe is made from a small, know sequence of DNA. The probe is complementary to the target DNA and can bind specifically to the targets that are anchored on the filter. This is known as hybridization and involves the filter being incubated with the single stranded DNA probe. That probe is either radioactively labeled (using <sup>32</sup>P) or marked with an enzyme. Excess probe is removed by extensive washing after the completion of the hybridization. | To identify the location (size) of a specific piece of DNA on the filter a sequence specific probe needs to be made. The probe is made from a small, know sequence of DNA. The probe is complementary to the target DNA and can bind specifically to the targets that are anchored on the filter. This is known as hybridization and involves the filter being incubated with the single stranded DNA probe. That probe is either radioactively labeled (using <sup>32</sup>P) or marked with an enzyme. Excess probe is removed by extensive washing after the completion of the hybridization. | ||
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==References== | ==References== | ||
<references/> | <references/>[[Category:Suggestion Bot Tag]] | ||
Latest revision as of 11:01, 20 October 2024
The Southern blot is a technique to detect the presence of a specific piece of DNA sequence. It is named after Edward M. Southern who developed the technique at Edinburgh University in the 1970 [1].
The Process
The goal of a Southern blot is to identify a specific fragment of DNA, often from a whole genome or complex mixture of DNA, using a sequence specific probe.
The DNA to be tested must be cut into pieces by a restriction enzyme that cleaves the DNA at specific sites in the DNA sequence. To sort the complex mixture of DNA fragments by size the mixture of DNA is passed through an agarose gel using an electrical current. The process is called gel electrophoresis; small pieces of DNA move rapidly through the gel than larger pieces.
The DNA trapped in the gel is treated with alkali to denature the DNA fragments to single stranded pieces. Then, by wicking liquid through the gel (blotting), the DNA is transferred to a nitrocellulose filter or charged nylon membrane. The DNA is attached to the filter permanently by either heating or crosslinking with UV light. Once attached to the filter the relative location of the DNA fragments from the agarose gel are preserved on the filter.
To identify the location (size) of a specific piece of DNA on the filter a sequence specific probe needs to be made. The probe is made from a small, know sequence of DNA. The probe is complementary to the target DNA and can bind specifically to the targets that are anchored on the filter. This is known as hybridization and involves the filter being incubated with the single stranded DNA probe. That probe is either radioactively labeled (using 32P) or marked with an enzyme. Excess probe is removed by extensive washing after the completion of the hybridization.
If the probe is labeled radioactively it can be detected with X-ray sensitive film, or more recently, fluorescent imagers have been used. If the label is an enzyme, a substrate is added that develops a color through interaction with the enzyme.[2][3].