Biotechnology and Recombinant DNA

Introduction

History

Cutting and Splicing

Inserting Recombinant
DNA Into Cells

Selecting Transformants

Applications I
Altering Phenotypes,
Making Products

Applications II
Study and
Characterization of DNA

Ethics

Inserting Recombinant DNA Molecules Into Cells

The most common ways to insert recombinant DNA into cells reflect the observations of how recombinant DNA forms in nature.

 

In bacteria, scientists had observed the passage of genetic material between organisms in three ways:

  • Conjugation - transfer of information using sex pili
  • Transduction - transfer of information by bacteriophages
  • Transformation - uptake of naked DNA from outside the cell

There are other ways, many based on advances in technology, but the main way foreign DNA is introduced into cells in the laboratory is by transformation and transduction.

 

Some of the other methods that may be used include protoplast fusion (which generates random recombination), microinjection into animal cells, and the use of "gene guns" to blast DNA-coated particles through cell walls and into plant cells (as we look at some lab tricks to enhance transformation you may find yourself wanting to classify the use of a gene gun as just a particularly violent form of transformation).

 

 

Microinjection

 

Gene Gun

 

Transformation

Transformation is the uptake of naked pieces of DNA from outsided the cell. Some bacteria are naturally competent to do this but others are not. The discovery that calcium chloride in the culture media could make bacteria take up plasmids greatly enhanced our ability to put selected recombinant DNA molecules into cells.

 

Another way to stimulate uptake of DNA by protoplasts and animal cells (no cell walls) is by passing an electric current through the culture medium. This causes pores to open up, thus the name electroporation.

 

Transduction

Infection is another way to get foreign DNA into cells. Genetically engineered bacteriophages or viruses can infect cells and introduce the desired recombinant DNA.

 

Vectors

Vectors are constructs that deliver the gene of interest to the host cells. There are a number of properties that are required to make a good vector:

  • Vectors should be self-replicating
  • Vectors should be small enough to manipulate outside the cell without breaking
  • Vectors should be protected from degradation by host cell enzymes. Circular plasmids are protected and viral vectors can insert into the host genome quickly to escape degradation.
  • Vectors should have selectable markers that will allow identification of host cells that have successfully taken the vector into the cell - there should also be a way to screen the vectors to insure the gene that was to be inserted is actually in the vector and that the host cells have not simply taken up re-annealed "blank" vectors.

Shuttle vectors are plasmids that can exist in several different bacterial species.

 

One can actually make combinations of plasmids and phage DNA and wrap them in phage coats to deliver larger pieces of DNA to bacteria. These constructs are called cosmids.

 

There are a number of other vectors used for cloning in different organisms. Yeast artificial chromosomes (YACs) have all the components necessary to conduct cloning studies in yeast and allow insertion of much larger (200 - 1500 kb) pieces of DNA.

 

Another vector used to clone eukaryotic DNA, bacterial artificial chromosomes (BACs) have been developed using the F factor (the fertility factor plasmid found in E. coli). BACs can hold fragments between 100 and 300 kb and are much more stable than YACs.

 

Plant viruses and animal viruses have also been engineered to deliver recombinant DNA molecules to eukaryotic cells. These vectors aim toward stable integration of the foreign DNA into the host genome.

Cloning

Let's look at the steps that would allow us to use a plasmid vector to clone a particular gene or create a DNA library.

 

First, cut the plasmid and the DNA that has the gene of interest with the same restriction enzyme.

  • Question: Where did we get the DNA that has the gene of interest?
  • Answer: In this case we got it from some organism that has the gene we want to work with. So we cracked open some cells, extracted the DNA (this is called genomic DNA), and digested it with the same restriction enzyme we will digest our plasmid with.

Second, mix the cut plasmid and digested DNA together and allow the restriction fragment to anneal with the cut plasmid.


Looks great, huh? But you know what happens when you do this. In the first place you don't mix one cut plasmid with all that DNA and think you'll get the one restriction fragment you want annealed to the plasmid.

 

You have many copies of plasmid, and they can all pick up any restriction fragment or they may simply reanneal with themselves.


 

Third, you take this mess of plasmids, some with restriction fragments and some without, and put them into cells (transformation).

 

As we said earlier, there are a couple of ways to get cells to take up plasmids (chemical treatment or electroporation) but remember, no process is 100% efficient.

 

This means after you've mixed cells and plasmids and stimulated uptake you'll need some way to determine whether individual cells are transformed and whether the transformants took up a recombinant plasmid or an "empty" plasmid that reannealed without incorporating a restriction fragment.

 

Once you figure out which cells are transformants with recombinant plasmids and "harvest" them you have what is known as a DNA library. A DNA library is a group of clones, each containing a restriction fragment, that represents the entire genome you digested with restriction fragments.

 

Of course you don't know which restriction fragment in which clone has which gene(s). Those genes of interest aren't painted red in real life, so you also need some way to determine whether or not a particular clone has the restriction fragment that contains your gene of interest.

 

This is called selection.

Introduction

History

Cutting and Splicing

Inserting Recombinant
DNA Into Cells

Selecting Transformants

Applications I

Applications II

Ethics