DNA & Biotechnology Notes—Teacher KEY
PreAP Biology
With the determination of DNA structure credited to Watson & Crick, and our subsequent understanding of its
primary functions in cell reproduction (DNA replication) & gene expression (we’ll talk about this later this semester),
scientists began to explore how this information could be exploited for the benefit of humanity. While some of the
innovations involving the human genome are shrouded in controversy, some, such as gel electrophoresis and polymerase
chain reaction (PCR), have been proven extremely valuable.
Old tools available for the diagnosis of genetic disorders include karyotypes, pedigrees, amniocentesis, chorionic
villi sampling, ultrasound, and blood tests. These tools are still used, but science has gone way beyond just diagnosing a
disorder! Scientists are looking toward treatments and cures, as well as enhancement and prevention of many genetic
traits in the Animal Kingdom as well as all 5 of the other kingdoms. We will talk more about these following our unit on
viruses and bacteria in the spring semester.
Gene technology really took off with the Human Genome Project. Initially a government venture with the goal of
sequencing the entire human genome (all 23 different chromosomes), it soon turned into a contest between a privately
funded team headed up by J. Craig Venter and the government team. The competition turned out to be just the spark
needed. The project wrapped up in 2003. Nature published a historic article on this contest (which I believe was called a
draw) in 2006. The methods used below are partially due to discoveries and innovations used during this historic quest
for knowledge.
Vocabulary Basics
Biotechnology – manipulation of organisms or their components to make useful products
Gene technology is a generic term covering many new techniques involving DNA and/or chromosomes. These new techniques include
genetic engineering, electrophoresis, Southern blots, DNA fingerprinting, and polymerase chain reaction (PCR).
Genetic engineering – direct manipulation of genes for practical purposes
Bioinformatics - application of computer science and information technology to the field of biology and medicine
Proteomics - term in the study of genetics which refers to all the proteins expressed by a genome
Genomics – study of whole sets of genes and their interactions once we have the entire genome sequence
Restriction enzymes—proteins that “cut” or break DNA molecules at certain internal positions.
Restriction Enzymes
The enzymes which break DNA molecules at internal positions are called restriction endonucleases. Enzymes that
degrade DNA by digesting the molecule from the ends of the DNA strand are termed exonucleases.
Restriction enzymes have developed into one of the primary tools in molecular biology. Each
restriction enzyme recognizes a specific nucleotide sequence. The enzyme “scans” the length of the DNA
molecule and then digests it at a particular recognition sequence. For example, the EcoRI endonuclease has the following
recognition sequence:
GAATTC
CTTAAG
It breaks the DNA at certain locations indicated by the dotted line
and produces “ragged ended” sequences often called “sticky ends”.
Other endonucleases cut the DNA cleanly at one specific base pair.
The diagram to the right shows 5 restriction enzymes and the
cuts they make within a strand of DNA. Using the diagram above,
determine the answers to the following questions:
1. Look at the DNA sequence that follows:
5’-TCGATATGGATCCGGGGAGAGAATTCATTATT-3’
3’-AGCTATACCTAGGCCCCTCTCTTAAGTAATAA-5’
Could it be cut by Alul? __NO__If so, show all sites where it could be cut using lines similar to the diagram at the
top of the page. What type of end does it produce? __blunt___
2. Look at the DNA sequence that follows:
5’-TCGATATGGATCCGGGGAGAGAATTCATTATT-3’
3’-AGCTATACCTAGGCCCCTCTCTTAAGTAATAA-5’
Could it be cut by EcoRI? _YES_ If so, show all sites where is could be cut. What type of end does it produce?
_sticky ends_
Restriction enzymes have many applications. For example, they are used in creating recombinant DNA/ transgenic
organisms, in gene splicing, and in gel electrophoresis. Transgenic organisms are organisms that have foreign DNA in
their cells. Gene splicing is when a gene from one organism is inserted into a chromosome or the genome of another
organism. Why would genes ever be spliced? They would be spliced for many applications, including altering phenotype
(make it glow), to give it resistance to a disease or an antibiotic, to make it produce a protein that it normally would not,
etc.
Electrophoresis
Electrophoresis is a tool used by many different kinds of scientists to separate molecules based on
characteristics such as molecular size, weight, or charge. The applications of electrophoresis in gene
technology involve separating pieces of DNA (that have been cut by restriction enzymes) by size and
making a Southern blot or a DNA fingerprint. Other techniques involve the separation of proteins using similar,
electrophoretic materials.
The DNA is put into a solution and squirted into a small well at one end of the gel (literally a piece of gelatin-like
material that is made of a seaweed extract plus other materials) in the electrophoresis chamber. The chamber is turned
on and a current passes through the chemicals inside. The DNA pieces will move toward the positive pole (because the
phosphates give DNA a negative charge). The small pieces will move the fastest.
(Here’s an easy way to think about why the small pieces move fastest. The gel is made of molecules that can get
in the way of the DNA moving. The smaller the piece, the easier it is to move past the gel molecules, just like it is easier
for you to walk alone between the trees in a dense forest than it is for you to walk between the trees with five friends all
holding hands.)
DNA Fingerprinting
DNA fingerprinting is so named because scientists have realized that a person’s DNA is as unique as
their fingerprint, therefore when restriction enzymes are used to cut a person’s DNA into pieces, the
number and size of the pieces is unique and creates a unique banding pattern on the electrophoresis gel.
The different pieces that result are called restriction fragment length polymorphisms (RFLPs). Polymorphism means
“many forms” and refers to the fact that the fragments are different lengths. What do RFLPs have to do with the use of
restriction enzymes in creating a DNA fingerprint using gel electrophoresis? RFLPs are the fragments of DNA that are
different lengths in different people because they contain the restriction sites for a particular enzyme (endonuclease) at
different places. They are important because this is what creates a unique DNA fingerprint for each person using gel
electrophoresis.
DNA fingerprinting is used to determine guilt or innocence (or more accurately, whether or not
someone was present where a crime occurred) using crime scene (forensic) material, as well as paternity,
identity, and even the presence or absence of a specific genotype, which can aid in the diagnosis of a
specific genetic disorder.
At a crime scene involving a murder, police recovered skin cells under the victim’s fingernails. The DNA in
the skin cells was analyzed- it was not the victim’s, and thus was presumed to be the assailant’s. Three
suspects were arrested for matching the description given by several witnesses. Their individual DNA was
analyzed, and the results are shown below. Can the correct suspect be determined?
Southern Blot
A Southern blot (named after the scientist who developed the technique) is used to identify specific sequences of
DNA that a scientist is interested in, such as determining which pieces have the insulin gene (p. 231, Figure 5).
DNA is run through an electrophoresis chamber and a piece of filter paper is laid on top of the gel to make an
exact copy of the banding pattern. The filter paper is soaked in a solution containing radioactive nucleic acid pieces that
are known to be complementary to the gene the scientist is looking for. The radioactive pieces will attach to the gene
sought and that band will glow, indicating that what you are looking for is present in that sample.
A little more – Southern blots can be used to detect and analyze differences in banding patterns – these differences result
from differences in DNA sequences on similar chromosomes from individual to individual or species to species. These
differences, which are cut at different places by restriction enzymes, are called restriction fragment length polymorphisms
or RFLPs (pronounced “rif-lips”)
Polymerase Chain Reaction (PCR)
Polymerase chain reaction is a method used to rapidly duplicate DNA segments – on the order of 1 billion copies
per hour! The process mimics DNA replication. First, high temperature is used to separate the strands of the DNA.
Then, low temperature and bacterial DNA polymerase are used to make copies (allow the free nucleotides in the solution
to copy the DNA strands that have been separated. The machine then cycles to high temperature again to start the
separation of the strands again. This continues over and over in a repeating cycle. Important applications include any
situation where you have limited DNA – crime scene material, diagnosing a genetic disorder, studying a fossil, etc.