Sequence Tagged Sites

by Georgina Cornwall, PhD

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    00:00 mutations are on chromosomes or if an individual has that particular mutation.

    00:01 Another thing we can use to identify pieces of DNA. So yes, we have chopped them up into pieces and we have perhaps sequenced them, but where are these pieces? How do we organize them? And we can determine how we can organize them by using sequence tag sites or STSs. Sequence tag sites can provide a sort of a scaffolding of how the pieces in the genome go together.

    00:27 We are getting a little bit more granular perhaps we got to Colorado. We are now looking at a map of the whole of the Colorado. We can't see the streets in Denver yet. When we look at sequence tag sites, we will be investigating locations of known DNA sequences on a chromosome. Now there are libraries of sequence tag sites, databases available online.

    00:52 The sequence tag sites are 200 to 500 base pairs long, pieces of sequence that have a single occurrence in the human genome. We have the sequence of the human genome and these are tag sites that come up only once and we know their specific location from previous research. DNA fragments from our DNA libraries can be cut up with restriction enzymes just like we have seen before. Common things here. They run through a gel with electrophoresis separating pieces by size as we have seen before. Now from each of the clones that we had in the libraries we have different pieces of DNA.

    01:32 And because of the sequence tag sites, we can align these pieces of DNA. One of the other pieces that come into here is polymerase chain reaction. You will recall. Previously we use polymerase chain reaction to amplify DNA in a PCR machine. We separate DNA and replicate DNA and stick it back together and replicate and stick it back together. Now we can identify these sequence tag sites using PCR and probes. The probes will attach when the DNA separates and then we can locate those probes by using visualization techniques.

    02:15 So we know whether that STS exists on that particular piece of DNA and we can put those pieces together to make a sequence. All of these DNA sequencing or physical mapping techniques really involved taking DNA from libraries chopping those chopped up pieces into more chopped up pieces and then trying to reassemble the puzzle once we have marked what is on them. Again here we are at the Colorado kind of level map looking at where Denver might be. STSs might tell us that sort of location. So now, let us think about how we can put these two pieces together. Often it will take a physical map and a genetic map and impose them upon each other to form a greater idea of where these genes are. We may know where the genes are based on phenotypes and recombination frequencies, but then we can actually use some of these markers that might be associated with the gene in order to figure out precisely how far away they are. The ultimate physical map is provided by the exact DNA sequence on a chromosome. Here we have moved from our state level map to our map of downtown Denver and specifically where a restaurant is that we want to visit. We can say exactly where the gene is on a chromosome using DNA sequencing. How do we do DNA sequencing? First of all, let us go back to the beginning of our technologies we were using restriction enzymes to cut up all of the DNA in some things genome. Let us say it is the human genome.

    04:00 And when we chop up all that DNA, we are going to store it in a DNA library and we have inserted them into plasmid factors. We ask these plasmid factors to be taken up by transformation and stored in bacteria. Here we have our volume of books. We call that a DNA library, it's not like a book library because each volume has a overlapping fragments. We have looked at few of the ways that we might line up these overlapping fragments, but now let us look at specifically how we sequence the DNA, then we will move into how we put these sequences back together some modes that are used for large genomes. Before we move in to how sequencing works.

    About the Lecture

    The lecture Sequence Tagged Sites by Georgina Cornwall, PhD is from the course Genomics.

    Included Quiz Questions

    1. sequenced-tagged sites.
    2. restriction fragment length polymorphisms.
    3. shotgun sequencing.
    4. consensus sequencing.
    5. clone by clone sequencing.
    1. STSs provide stability to the physical structure of the Taq polymerase enzyme at high-temperature conditions of the PCR.
    2. A sequence-tagged site is composed of a short DNA sequence of 200-500 bp which occurs for a single time in the genome.
    3. Specific primers are used in a polymerase chain reaction to locating the STSs.
    4. STSs serve as landmarks in the development of physical maps of a genome.
    5. STSs are used for the detection of microdeletions in Azoospermia (AZF) genes in infertile men.

    Author of lecture Sequence Tagged Sites

     Georgina Cornwall, PhD

    Georgina Cornwall, PhD

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