Restriction Mapping

by Georgina Cornwall, PhD

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    00:01 In these next two lectures, we will be embarking on the study of genomics, which is the study of what the genome does. What does it code for? Before we can really think about what the parts do, we need to map the genome and sequence the genome. In this first lecture, we will learn to distinguish between genetic and physical maps as well as describe the process of DNA sequencing and finally you will be able to characterize two different methods for sequencing genomes, the clone-by-clone method versus the shotgun method.

    00:39 Let us begin by thinking about genetic versus physical maps. Previously we have examined genetic mapping by looking at recombination frequencies and predicting the relative distance of genes. How far genes are apart on a chromosome? This is okay, but it does not give us a very close map. Physical mapping allows us to find the actual physical location of genes on the chromosomes like all the way down such a granular level. We know precisely at what letter a gene begins. Much like we would have a map if you were trying to go to a strange place, you have a map with a fairly broad spectrum and we are going to increase resolution.

    01:25 You are looking at google maps say and you will look at the whole state and you decide that you want to go to Denver and so you can see Denver relative to where you are. And you drive into Denver and you want to get a little bit more granular. We zoom in further and zoom in further and eventually you are down there on downtown Denver and you are looking for a specific restaurant. We have a very fine resolution map now showing you specifically where you are and where the door to that restaurant is. You can even get street if you like. Looking at the gene sequence or the DNA sequence can give us that granular view of exactly where specific genes are in the genome. We are going to revisit a lot of technologies that we have previously visited. We can use restriction enzyme sites in order to cut DNA into fragments and try to put together physical maps. We could probably have our DNA from a DNA library could be a CDNA library and the segment of DNA can be cut with a variety of different restriction enzymes. We have seen these before. Then we run those fragments through our gels in different lanes, at the same time, we put the enzyme A in one lane and the enzyme B solution in the other lane and then a combination of the two in the third lane. We'd run them together. The negatively charged DNA runs towards the positive pole and the fragments separate by size. Larger fragments do they go further or less far? Larger fragments have a harder time making it through the gel so they will go less distance through the gel while smaller fragments will go further distances through the gel.

    03:16 Then we can take all of these pieces and compare the sizes and lengths and we have perhaps marked it with some markers whether it is dyes or tags. We will look at some specifics of those shortly and we can piece these fragments together that we have obtained from our DNA library and decide what the sequence is on the chromosome. This is a sort of an overview of how we make genetic maps. But in order to really make them, we have to have some sort of labelling and tags.

    About the Lecture

    The lecture Restriction Mapping by Georgina Cornwall, PhD is from the course Genomics.

    Included Quiz Questions

    1. Restriction mapping provides an imaginary location of the genes on the centromeres.
    2. A map of known restriction sites within a particular sequence of DNA, is called as a restriction map for that DNA molecule.
    3. The single and double restriction digestion techniques help in finding the relative position of restriction sites on the plasmid.
    4. The orientation of an insert in the cloning vector can be determined by using restriction mapping techniques.
    5. The restriction enzymes play a crucial role in the construction of restriction maps.
    1. …gives information regarding the actual physical location of genes on the DNA molecule.
    2. …gives information regarding the location of mRNA in the cytoplasm.
    3. …gives information regarding the location of ribosomes on the surface of RER.
    4. …gives the relative position of the genes based on recombination frequency.
    5. …gives the relative activity of the genes based on transcription rates.

    Author of lecture Restriction Mapping

     Georgina Cornwall, PhD

    Georgina Cornwall, PhD

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