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Instruction 5-4

Structure and Function of DNA, RNA | Base Pairing Rules | Genetic Engineering | DNA Technology and Recombinant DNA | Adding DNA to Bacterial Genetic Material

DNA Technology and Recombinant DNA
CA Biology GR. 9-12. 5.d.

To understand the significance and applications of biotechnology, one needs a basic understanding of rDNA (recombinant DNA) genetically engineered DNA made by recombining fragments of DNA that's been obtained from other organisms.

We have told you about DNA and genetic engineering in several Instructions now, but it's such an important subject that we think that it's important to review it again.

One way to think about it is to use the analogy of language. The Language of Recombinant DNA.

We've told you about nucleotide bases (A, T, G and C), about codons and about genes. Using the analogy of language, here's what each would be:

  • The nucleotide bases would be letters,
     
  • The codons would be words,
     
  • The genes would be sentences,
     
  • DNA would be a book (the instruction book of life).

As you know, nucleotide bases by themselves convey no message. Instead, they act in strings of three called codons.

The function of the codons on the DNA molecule is to give instructions for specifying and ordering amino acids. And amino acids, of course, are the structural elements of proteins, which in turn are the basic biochemical units that drive all biological processes.

There are only twenty amino acids found in proteins - and the codes for ordering them are universal. This means that the sequence of nucleotide letters to specify amino acid words is the same for everything that has ever lived - from tree sloths to Tyrannosaurus Rex (and us). But these amino acid words can be combined in many different ways to make thousands of different protein sentences.

Certain proteins, called enzymes, are catalysts (agents that cause change but aren't themselves changed during a chemical process). Others, called structural proteins, help build cells and tissues.

As you know, one codon contains the instructions for one amino acid and sequences of codons specify the production of proteins.

Groups of codons that have been arranged in sentences to form specific proteins are called "genes".

And finally, it's in the genes (the blueprints for making proteins) that DNA actually does something -it writes a book.

Through a complicated series of biochemical processes, the instructions contained in the genes are translated into the actual stuff that organisms are made of - which is where rDNA technology comes in.

Returning to our analogy of DNA as a language with letters, words and sentences, one can think of genetic engineering as editing the DNA book to produce a desired result.

 

The Editing Process

The actual editing - which is called the "insertion process" - involves manipulating incredibly tiny pieces of incredibly tiny organisms.

Basically, however, it is still "cutting and pasting" - using restriction enzymes as the scissors and DNA ligase as the paste.

There are over a hundred different restriction enzymes (also called restriction endonucleases) - each of which cuts a specific base sequence of the DNA molecule. When these "scissors" are used singly or in the right combination, the correct segment of the DNA molecule can be isolated.

Once isolated, the segment is "cut" and "pasted" into plasmid DNA, a special kind of circular DNA that is frequently used as a vehicle for editing (and which we'll tell you more about in our next Instruction).

Fragments of bacterial DNA exist that may be integrated into the plasmid chromosome, but only at specific locations.

Let's take a look at the production of genetically engineered insulin as an example of how it all works.

The Production of Insulin


To create genetically engineered insulin for human use, scientists cut the "sentence" (the gene for insulin production in humans) and paste it into the DNA of Escherichia coli (E. coli), a bacterium that inhabits the human digestive tract.

E. coli cells divide very rapidly making billions of copies of themselves, with each bacterium carrying in its DNA a faithful replica of the gene for insulin production. Even more importantly, each new E. coli cell will inherit the human insulin gene "sentence."

The genetic engineering of insulin has been a great success - saving the lives of countless diabetics.

There are also many other explorations under way concerning the diagnosis, treatment and possible cure for other genetically related diseases.

One promising area of exploration is called Cytogenics.


Cytogenics 

Cytogenetics is the study of chromosomes and of conditions caused by an abnormal number of chromosomes or an altered chromosome structure.

As you know, in humans, diploid somatic (non-sex) cells normally contain 46 chromosomes.

A chromosome number which is an exact multiple of the haploid number (23 in gametes) and exceeds the diploid number (46) is called a polyploidy. This is a condition that results when an individual gains a full set of extra chromosomes. Another name for this condition is N4.

A chromosome number that is not an exact multiple of the haploid number is called an aneuploidy.


Polyploidy and Aneuploidy

Polyploidy can arise either when an egg is fertilized by two sperm (dispermy) or from the failure of one of the maturation divisions of the egg or the sperm, which produces a diploid gamete.

A triploid fetus would be 69,XXY (the most common), 69,XXX or 69,XYY depending on the origin of the extra set of chromosomes.

Four times the haploid number is called tetraploidy and is usually due to the failure of the completion of the first zygotic division.

Aneuploidy arises from failure of paired chromosomes or sister chromatids to disjoin at anaphase (non-disjunction). It may also be due to delayed movement of a chromosome at anaphase (anaphase lag). This produces two cells with the following aberrations -- one with an extra copy of a chromosome (trisomy) and one with a missing copy of that chromosome (monosomy).

Structural Aberrations

Structural aberrations come about as a result of chromosome breakage.

When a chromosome breaks, two unstable sticky ends are produced. Generally, repair mechanisms rejoin these two ends without delay. However, if more than one break has occurred, repair mechanisms may not be able to distinguish one sticky end from another and may rejoin the wrong ends.

Here are the most common chromosomal structural aberrations:

  • Deletions - these involve the loss of material from a single chromosome. The effects are typically severe since there is a loss of genetic material.
     
  • Inversions- these occur when there are two breaks within a single chromosome and the broken segment slips 180 (inverts) and reattaches to form a chromosome that is structurally out-of-sequence.
     
  • Translocations - these involve the exchange of material between two or more chromosomes. Gene expression is
    usually unaffected by translocation. However, unequal crossing-over in reciprocal recombination can occur as a result of mispairing the identical duplicated gene sequences.

Numerical and structural abnormalities can be further divided into two main categories.

These categories are:

  • Constitutional - those an organism is born with
     
  • Acquired - those that arise as secondary changes to other diseases such as cancer

Sometimes individuals are found who have both normal and abnormal cell lines. These people are called mosaics and in the vast majority of these cases the abnormal cell line has a numerical chromosome abnormality.

Structural mosaics are extremely rare and the degree to which an individual is clinically affected usually depends on the percentage of abnormal cells involved.

Video Instruction
*Availability of You Tube video links may vary. eTAP has no control of these materials.

 

Experiments for Home and Classroom

The identification and location of DNA segments is the critical first step in genetic engineering. In these two activities from the University of Utah Learning Center, students are invited to learn how to measure and analyze DNA segments just as they would in a professional genetics laboratory. The first activity is in a process called Gel Electrophoresis.
http://gslc.genetics.utah.edu/units/biotech/gel

The second activity is in DNA Microarray Analysis -- one of the fastest-growing new technologies in the field of genetic research. Scientists are using DNA microarrays to investigate everything from cancer to pest control. Learn how to find the differences between a healthy cell and a cancer cell.
http://gslc.genetics.utah.edu/units/biotech/microarray 

In this interactive activity, called "Welcome to iDNAfication," students are invited to use the principles of molecular biology to solve a mystery. Teachers are invited to customize the game for their students by e-mailing SWBIC (the Southwest Biotechnology and Informatics Center at New Mexico State University).
http://www.swbic.org/cgi-bin/idna.cgi 

 

Reading List
 
Mean Genes
by
Terry Burnham and Jay Phelan
 

for Students, Parents and Teachers

Now let's do Practice Exercise 5-4 (top).

Next Page: Adding DNA to Bacterial Genetic Material (top)