MOL - Gene Technology [LESSON]

Gene Technology

Many examples of modern biotechnology depend on the ability to analyze, manipulate, and cut and paste pieces of DNA. There are numerous applications for these techniques, from gene therapies that can relieve symptoms of genetic disorders to cloning organisms. Let’s go over the basics of some of the most common techniques.

Watch the Molecular Biology video below for a great introduction to these concepts.

Cutting and Pasting DNA

A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target sequence and cuts DNA into two pieces at or near that site.  Restriction enzymes are found naturally in many prokaryotic cells to serve as a protection against viral DNA insertion. Many restriction enzymes produce cut ends with short, single-stranded overhangs (sticky ends). If two molecules have matching sticky ends, they can base-pair and stick together. However, they won't combine to form an unbroken DNA molecule until they are joined by DNA ligase, which seals gaps in the DNA backbone.

One of the most common restriction enzymes is called EcoRI, shown below. It recognizes a particular sequence and cuts through the DNA, leaving sticky ends.

EcoRI cutting DNA at its target sequence

Once the target DNA is cut, it can be inserted into another sequence of DNA that has been cut by the same restriction enzyme. Then, the gaps between strands are sealed with DNA ligase.

The image shows the ligase sealing the gaps in the backbone between segments of DNA.

Often, the receiving DNA is that of a plasmid, a small circular loop of DNA in prokaryotes that is separate from the chromosomal DNA. Plasmids are useful for storing genes of interest.

The image shows the plasmid being opened and the target sequence inserted.

Once they are joined by ligase, the fragments become a single piece of unbroken DNA. The target gene has now been inserted into the plasmid, making a recombinant plasmid.

The recombinant plasmid contains the target gene that has been transferred into the original plasmid.

Recently, a system of cutting at ANY target sequence has been discovered. The Crispr-Cas9 system has revolutionized gene technology in the past decade. 

Watch the CRISPR video below to learn more about this system.

Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) is a technique used to make mass quantities of a gene of interest. Think of it like a DNA copy machine. We need to include the following things in the reaction tube:

  • Taq polymerase – a special type of DNA polymerase enzyme isolated from a heat-tolerant bacteria which can withstand high temperatures.
  • DNA nucleotides – to build the new strands with.
  • Primer – short sequences of DNA that bind just outside the target DNA to copy.

 

Template DNA Primers bind to the template Taq polymerase extends primers.

PCR machines run a cycle where they heat and cool.  First, they heat up the target DNA so it denatures. Then, it cools so that the primer can bind and Taq polymerase can add new nucleotides to form new copies of the target DNA. This process repeats and we see an exponential increase in the target DNA.

PCR machines run a cycle where they heat and cool. 

Now, there is plenty of DNA to use for other purposes in the laboratory!

Gel Electrophoresis

Gel electrophoresis is a technique in which fragments of DNA are pulled through a gel matrix by an electric current, and it separates DNA fragments according to size. A standard, or DNA ladder, is typically included so that the size of the fragments in the PCR sample can be determined.

Gels for DNA separation are often made out of a polysaccharide called agarose, which comes as dry, powdered flakes. When the agarose is heated in a buffer (water with some salts in it) and allowed to cool, it will form a solid, slightly squishy gel. At the molecular level, the gel is a matrix of agarose molecules that are held together by hydrogen bonds and form tiny pores. At one end, the gel has pocket-like indentations called wells, which are where the DNA samples will be placed. Before the DNA samples are added, the gel must be placed in a gel box. One end of the box is hooked to a positive electrode, while the other end is hooked to a negative electrode. The main body of the box, where the gel is placed, is filled with a salt-containing buffer solution that can conduct current.

At one end, the gel has pocket-like indentations called wells, which are where the DNA samples will be placed.

As the gel runs, shorter pieces of DNA will travel through the pores of the gel matrix faster than longer ones. After the gel has run for typically about an hour, the shortest pieces of DNA will be close to the positive end of the gel, while the longest pieces of DNA will remain near the wells.

Watch the Gel video below.

Try the Electrophoresis Questions below.

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