DNA Libraries

by Georgina Cornwall, PhD

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    00:01 Welcome back to our second lecture on biotechnology. In this lecture, we will apply some of the techniques that we learned in the first lecture and explore how DNA libraries are put together as well as examine some DNA analysis techniques. And finally we will look at some applications of biotechnology in medicine and agriculture. So by the end of this lecture, you should be able to explain the benefit of DNA libraries as well as compare RFLP and STR in DNA fingerprinting.

    00:35 What are those you may ask? Well stay with me and you will find out. By the end of this lecture, you will also be able to discuss some of the applications of biotechnology in both agriculture and medicine. Let us move into the beginning of genomic libraries. Genomic libraries are similar to our libraries except that there are volumes of books that have overlapping sequences. They are not independent volumes. There is often some repeats in a DNA library. They contain a representation of the entire genome of an organism. The whole point is to store that genome and fragments so that we can maybe sequence it or figure out some of the genes in the genome too. When we think about storing these fragments in a library essentially all we are doing is taking DNA fragments from an organism's DNA workshopping them with restriction enzymes. And then we will insert them into a plasmid vector. We already know how that works from the previous lecture and then we take those vectors and ask bacteria to transform and pick up these vectors and store them for us in a nice neat library. Each piece of DNA is in a different bacteria and there are perhaps hundreds of bacteria to represent one genome of maybe the fruit fly. Keep it mind, plasma DNA is fairly small when we think about bacterial plasmids. Artificial chromosomes have been used to store larger fragments, but we will move on to that later.

    02:13 How do we get copies of eukaryotic genes? This is really a good question because if you recall from looking at our DNA transcription and translation, how genes are expressed, you might remember that some portions of the DNA are expressed and some portions are not in the form of introns and exons. The exons are expressed, but bacteria don't have the machinery to clip out the intron, the unexpressed pieces. So we have to trick them into picking up just the transcribable copies of DNA and this is the way we do it by making a complimentary DNA library. First of all, we will take the eukaryotic DNA template and then we have transcription occur. You know how that works by now. And by that, we get a primary mRNA transcript and then we need to clip out the introns in order to just have the expressed sequence where we see it in the mature messenger RNA. Now that we have this mature messenger RNA, we have the sequence of RNA that codes only for the expressed portions of the proteins.

    03:28 And in order to trick the bacteria to take in all we need and just what we need, we need to get a DNA copy of this. That is where the complimentary DNA comes in. We will introduce an enzyme called reverse transcriptase, which guess what. Transcribes DNA from messenger RNA, regular transcriptase goes forward. Reverse transcriptase, we are taking a messenger RNA molecule making a complimentary DNA strand. Then we have single stranded complimentary DNA and we need to get rid of the messenger RNA template. So we will degrade those enzymes and the messenger RNA will disappear and now we need to introduce the DNA polymerase, the enzyme that is going to polymerize the complimentary strands. So DNA polymerase makes the complimentary strand and now we have double-stranded complimentary DNA with no introns ready to express the entire protein. We can then introduce that into our DNA library.

    About the Lecture

    The lecture DNA Libraries by Georgina Cornwall, PhD is from the course Biotechnology.

    Included Quiz Questions

    1. Reverse transcriptase and DNA Polymerase
    2. Reverse transcriptase and Taq polymerase
    3. DNA ligase and Taq polymerase
    4. Taq polymerase and RNA polymerase
    1. Exonuclease — to create the genome fragments during cDNA library
    2. Genomic library — a representation of entire genome of an organism
    3. Reverse transcriptase — synthesis of cDNA from mRNA
    4. DNA ligase — to ligate the genome fragments into the cloning vector
    5. cDNA library — a representation of coding regions of expressed genes of an organism

    Author of lecture DNA Libraries

     Georgina Cornwall, PhD

    Georgina Cornwall, PhD

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