DNA Technology

Recombinant DNA technology refers to the techniques used to transplant genes from one living source into another where it will be expressed.

 

How Does it work?:

The science of DNA technology includes the use of special enzymes called restriction enzymes, DNA vectors, and the host organisms. We will take a look at each of these groups in the following section.

 

Restriction Enzymes:

These special enzymes were discovered in the late 1960's as naturally occurring agents in bacteria. They protect the bacterium against foreign DNA from other organisms. Invading DNA is cut into pieces and made inoperable. This process is called restriction. As with any enzyme, these are specific in the job they do. Many of them only recognize short, specific nucleotide sequences( recognition sequences) and cut at specific points within those sequences. Bacteria protect their own DNA by a process called methylation. During this process methyl groups are added to the nucleotides within the recognition sequence. There are several hundred restriction enzymes and about 100 different recognition sequences.

Recognition Sequences: are symmetric in that the same sequence of 4 to 8 nucleotides is found on both strands, but run in opposite directions. The restriction enzymes usually cut the phosphodiester bonds of both strands in a staggered manner. The result being both ends have a single stranded area called the sticky ends. It is within this space that the new piece of DNA is added, attaching to the sticky ends. See the diagram below.

 

 

These unions are temporary until the enzyme DNA ligase is added to catalyze the formation of the phosphodiester bonds.

Vectors: are used as carriers for moving DNA from test tubes into cells. Bacterial plasmids and viruses are the most widely used vectors in DNA transfer. Bacterial cells can pick up the DNA through the process of transformation. Lambda phages are used by eliminating the middle of its liner genome and adding the foreign DNA in the created space. The phage is then introduced into the bacterial cell where it replicated itself via the lysogenic cycle.

Host Organisms: Bacteria are usually used as hosts in genetic engineering. There are several reasons why they are chosen. 1. Bacterial DNA can be easily isolated and reintroduced into bacterial cells. 2. Bacterial cultures grow quickly. Some disadvantages surface as well: 1. Bacteria, being prokaryotic, may not be able to use the information in eukaryotic genes. 2. Bacterial cells cannot make the the necessary changes in transcription to produce some eukaryotic proteins. Eukaryotic cells can also be used as hosts. Yeast cells and some plant and animal cells can be a host for foreign DNA, but it is often difficult to get such cells to take up engineered DNA.

Steps for using Bacteria and Plasmids to Clone Genes:

1. Isolation of two kinds of DNA.

2. Treatment of plasmid and foreign DNA with the same restriction enzyme.

3. Mixture of foreign DNA with clipped plasmids.

4. Addition of DNA ligase.

5. Introduction of recombinant plasmid into bacterial cells.

6. Production of multiple gene copies by gene cloning and selection process for transformed cells.

7. Final screening for transformed cells.

 

 

Additional Methods for Analyzing and Cloning Nucleotide Sequences:

1. Gel Electrophoresis. is used to separate either nucleic acids or proteins based upon molecular size, charge and other physical properties. Various DNA's can be identified by their characteristic banding patterns after being cut with their particular retraction enzymes. Genes can be isolated, purified and recovered from the gel with full biological activity.

2. DNA Synthesis. It is possible to create known short DNA sequences in the laboratory. The Sanger Method is used to identify the sequence of a DNA molecule. This method is based on: a). use of restriction enzymes into small reproducible fragments. b). produce in a test tube DNA strands complimentary to a strand from the restricted fragment. c). Add a modified nucleotide that blocks further DNA synthesis.

3. The Polymerase Chain Reaction. This is another process that will allow DNA to be copied and amss produced in vitro. This process needs only a small bit of DNA and is being used in reconstruction of ancient DNA, minute specimens from crime secenes, DNA from cells infected with hard to find diseases, and DNA from single embryonic cells for prenatal diagnosis.

4. Hybridization. is used to determine the presence of a specific nucleotide sequence. Labeled probes complementary to the gene of intrest are allowed to bind to DNA from the cells being tested. This will determine if the gene being sought is present. It will also account for the numbers of these sequences, the size of the restriction fragment, and determine if the gene is made of mRNA, and how much of the mRNA is present.

Much of the above technology is being practiced in The Human Genome Project. This is an enormous effort to map and sequence the DNA of the human genome. Click here for a link to this project. Genome update article.

 

 

To practice this type of technology click here to link up with AP Lab Number 6 Molecular Biology.