Course: SAT 170 Introductory Biotechnology

Unit 7 Learning Module

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DNA Technology Part 2
In our last unit on DNA technology we spent a lot of time explaining the processes used to recombine, separate, clone and sequence DNA. In this unit we will explain the biotechnology methods that focus on how we can detect and store specific pieces of DNA. We will also briefly consider some of the applications of DNA technology including the development of genetically modified organisms (GMO’s), gene therapy and protein therapy.

An organism’s genome is comprised of their entire collection of DNA organized into chromosomes. This includes their genes, as well as their non-coding DNA (shown here as intergenic sequences).

Storage and Detection of DNA

DNA libraries are often very useful for studying DNA. A DNA library consists of a collection of DNA fragments that can be isolated and cloned for further study. There are two main types of DNA libraries: genomic libraries and cDNA libraries.

A genomic library represents a collection of DNA from a particular organism. In many cases, a DNA library will hold that organisms complete genome. The DNA from this organism is divided into multiple fragments using restriction enzymes. Each fragment is then stored in a cloning vector which has been cut with the same restriction enzyme. This vector can be used at any point in time to introduce this DNA fragment into a host cell which will create copies of that DNA fragment. There are many types of cloning vectors that are utilized when constructing a genomic library. Each vector is capable of carrying a certain amount of DNA nucleotides and each has specific limitations and advantages. The choice of vector will depend on the characteristics and purpose of the genomic library. The most important features of a cloning vector include the size of DNA fragments that they are able to hold, whether or not they contain an origin of replication, an RNA polymerase promoter sequence and a selectable marker gene. The size of a particular vector will determine the amount of DNA that it is able to hold; these sizes are often reported in thousands of nucleotide bases known as a kilobase (kb).  The origin of replication, as discussed in unit 3, is the sequence where helicase will bind to begin the process of DNA replication. If a vector is going to be useful, it needs to have an origin of replication so that it can be copied once put into a host cell. Likewise, without an RNA polymerase promoter sequence the DNA can never be expressed into a functional protein. Recall from unit 4, the promoter serves as the site for RNA polymerase binding during the transcription step of gene expression. In order to determine whether or not a host cell has actually taken in the cloning vector, selectable marker genes are used. Such markers can be detected or measured (such as a fluorescent signal) as an indicator that the host cell has successfully incorporated the vector for replication and/or expression of the DNA in that vector.

This image illustrates the basic steps of the formation of a cDNA library

Plasmids are commonly used vectors because of their relative ease of isolation and manipulation. However, the plasmid vectors are limited to a maximum of around 12 kb and can only be incorporated for replication and expression in bacterial host cells.

Bacterial Artificial Chromosomes (BACs) have the same limitation of plasmids in that they can only be incorporated for replication and expression in bacterial host cells, but they are capable of holding a much larger amount of DNA. BACs typically have a maximum size of around 300 kb.

To overcome the problems of using bacteria, which are prokaryotes for host cells, Yeast Artificial Chromosomes (YACs) are often used. These vectors are capable of carrying 200-2000 kb but can only be incorporated for replication and expression in yeast host cells.

Human Artificial Chromosomes (HACs) are a relatively new development in the field of genomic cloning. Such chromosomes are capable of holding several thousand kilobases of nucleotides (which is why their size capacity is often reported in megabases) and can be grown in human cells.

Since most organisms carry a lot of DNA that does not code for particular proteins, known as non-coding, researchers will use cDNA libraries to evaluate the genes, or coding DNA, of that organism. To construct a cDNA library, the mRNA is isolated from a particular organism. This mRNA can then be converted to a complementary double stranded DNA molecule. Such complementary DNA is known as cDNA. The enzyme that creates the cDNA using the mRNA as a template is known as reverse transcriptase (RT); named as such because it carries out a reaction that is essentially the reverse of the normal process of transcription. Recall from unit 4 that transcription, which is carried out by RNA polymerase, copies a DNA molecule into a complementary mRNA. This process of reverse transcription occurs naturally in a type of virus known as the retrovirus. The RT enzyme was isolated from such retroviruses. Once the cDNA molecules are created, they can be stored in a library much like the genomic libraries previously discussed.