Biotechnology

BIO 101

Life Science

Dr. D. L. Daley

What is Biotechnology?

Any use of organisms, cells or biological molecules to achieve practical goals.

For over 10,000 years humans have used yeast cells to produce bread, beer and wine

Selective breed of plants and animals is also about 10,000 years old

Genetic Engineering

Genetically engineered cells or organisms may have had genes deleted, added or changed

The intent of genetic engineering is to learn more about how cells and genes work, to develop treatment for diseases, produce biologically important molecules and to improve the characteristics of plants and animals used in agriculture

Vast quantities of recombined DNA can be grown in organisms like bacteria, viruses and yeast cells and then transferred to other organisms

Plants and animals that express modified DNA are called transgenic or genetically modified organisms (GMOs)

Recombinant DNA Technology

Dramatic growth in this area of biology since the 1970’s

This technology is routinely used in many fields of biological research

Pharmaceutical industry commonly produces its products with recombinant DNA technology

Insulin is now produced this way as are some vaccines like that against hepatitis B

Forensic science use this technology in DNA fingerprinting and medicine uses recombined DNA technology in the diagnosis of inherited disorders

DNA Recombination in Nature

Sexual reproduction

Transformation - a process where bacteria naturally pickup fragments of DNA (chromosomes) or plasmids (tiny circular pieces of DNA that may carry useful genes)

These plasmids are commonly released into the environment when a bacterium dies and may be picked up by another bacterium

DNA Recombination in Nature

Viruses may transfer DNA when they infect their host’s cells

Biotechnology and Forensic Science

Forensic investigators need to identify victims and criminals and samples of cheek cells, hair follicles or semen may be old or limited

Two techniques are commonly used to match DNA samples

First the often small samples of DNA are amplified so that enough DNA is available for analysis

Next they determine whether the various DNA samples match

Amplifying DNA

Polymerase chain reaction (PCR) - developed by Kary Mullins of Cetus Corporation in 1986

Shared Nobel prize in chemistry in 1993 for his work on PCR

PCR produces unlimited amounts of DNA and can be used to amplify selected pieces of DNA

To start DNA replication, the enzyme DNA polymerase needs to be shown where to start to copy a piece of DNA - A primer, a small piece of complementary RNA is attached with enzymes to the place where DNA replication is to begin

In PCR, the nucleotide sequence of the beginning and end of the DNA to be amplified must be known

Amplifying DNA

In a test tube, the DNA is mixed with primers, free nucleotides and a special DNA polymerase that comes from bacteria that live in hot springs

The PCR process

1. Test tube heated to 194-203°F (90-95°C) - Breaks hydrogen bonds - yields single stranded DNA

2. Temp is lowered to 122°F (50°C) - the two different primers can now hydrogen bond to original DNA fragments

3. Temp is now raised to 158-161.6°F (70-72°C) - DNA polymerase directed by the primers now uses free nucleotides to make copies of the DNA segment bounded by the primers

4. This cycle is repeated as many times as desired - 20 PCR cycles can make about a million copies of the original DNA (PCR is a geometric progression - 1®2®4®8®16®32®64®128®256®512®1,024®2,048®4,096®8,192®16,384®32768®65,536®131,072®262,144®524,288®1,048,526)

PCR

Choice of Primers in Forensics

In forensics, scientists have found that small repeating pieces of DNA, called short tandem repeats (STRs) can be used to identify people with astonishing accuracy

STRs are often described as short stuttering genes

Each STR is only 2 to 5 nucleotides repeated about 5 to 15 times right next to each other

As with any gene, different people may have different alleles of the STRs - For STRs each allele is simply a different number of repeats of the same few nucleotides

Choice of Primers in Forensics

In 1999, British and American law enforcement agencies agreed to use a set of 10 to 13 STR, each 4 nucleotides long that vary greatly among individuals

A perfect match of 10 STRs in a suspects DNA and that found at the crime scene means there is less than one chance in a trillion that the two DNA samples did not come from the same person

Also the DNA around the STRs doesn’t seem to degrade very fast, so even old DNA samples usually have STRs intact

Forensics labs only use PCR primers that amplify the DNA immediately around the STR’s

STR alleles vary in how many times they repeat and thus in size

An STR with more repeats has more nucleotides and is larger and therefore forensics labs must be able to identify each STR in a DNA sample and determine its size

Gel Electrophoresis

Mixtures of DNA pieces can be separated by a technique called gel electrophoresis

The mixture of DNA pieces is loaded into shallow grooves, called wells in a slab of agarose, a carbohydrate from seaweed

The gel represents a meshwork of carbohydrate fibers

When the gel is placed in a bath of buffer and electrodes at each end so that current can be passed through the gel

Gel Electrophoresis

The phosphate group of nucleotides is negatively charged and when a current is passed through the gel the DNA fragments move away from the negative electrode and toward the positive electrode

Smaller fragments of DNA move more easily through the holes in the fibers of the agarose gel and thus move more rapidly toward the positive electrode

Eventually the DNA fragments are separated by size, forming distinct bands on the gel

DNA Probes Label Specific Sequences

Unfortunately DNA bands are invisible in the gel

There are several dyes that can stain the bands of DNA but are not used commonly in forensics or medicine

The problem is that there may be many DNA fragments of the approximately same size

Eg. 5 or 6 STRs with the same number of repeats might be mixed together in the same band

Therefore how can you identify a specific STR? Answer - complimentary base pairing!

DNA probes are single stranded pieces of DNA that are complimentary to a given STR

The DNA probes are labeled either with radioactivity or one of several colored molecules

DNA Probes Label Specific Sequences

When the gel is finished running the DNA from the invisible bars is next transferred to nylon paper via electrical current.

The nylon paper is bathed in a solution of labeled DNA probes that are complimentary to the specific DNA segments in the original DNA

In modern forensics labs the STRs are usually labeled directly with colored molecules during the PCR reaction

Biotechnology and Agriculture

The goals of agriculture are to grow as much food as possible, for as little money as possible with minimal loss from pests like insects and weeds.

As of 2005, 52% of corn, 79% of cotton and 87% of soybean grown in the US were transgenic (contain genes from other species)

To promote insect resistance many crops have been given a gene called Bt (from a bacterium) - this gene codes for a protein that damages the digestive tract of insects and not mammals

Getting the Bt Gene Into Cotton Plants

First the gene is isolated from the organism that makes it or it is synthesized in the lab via PCR or DNA synthesizers

The gene is then inserted into a plasmid and many copies of it can now be produced

Restriction Enzymes

Restriction enzymes are used to insert genes into plasmids

The restriction enzyme cuts DNA at a specific nucleotide sequence

If the same restriction enzyme is used to cut the Bt gene and plasmid - the gene can easily be inserted into the plasmid - the bases will easily pair-up

A ligase enzyme seals up the plasmid DNA with the new Bt gene

Bacterial Plasmids Insert Genes Into Plant Cells

Next the plant cells are infected with the transgenic bacterium

In certain bacteria the plasmids easily insert their DNA into the plants cell’s chromosomes

Appropriate hormone treatment stimulates the transgenic plants cells to divide and differentiate into an entire plant

These plants are bred to one another to create commercially valuable plants the resist insect attack

Biotechnology and the Human Genome

Human Genome Project (launched in 1990) - goal was to determine the nucleotide sequence of all of the DNA in our entire set of gene, the human genome

By 2003 molecular biologist from around the world had sequenced the human genome with an accuracy of 99.99%

The human genome contains about 21,000 genes which is only 2% of the total DNA

Some of the other 98% consists of promoters and regions that regulate how often each gene is transcribed - but it is not really known what most of the DNA does

Value of the Knowing the Sequence of the Human Genome

1. Many new genes were discovered that were previously unknown - knowing the nucleotide sequence allows molecular biologist to work backwards and figure out what the genes do

2. Knowing the sequence of nucleotides will have an enormous impact on the practice of medicine in the future

In 1990 only about 100 genes were know to be associated with disease - now it is over 1400 genes know to be associated with disease

Value of the Knowing the Sequence of the Human Genome

3. There is no single “human genome” (we would all be identical twins) - most of the DNA of everyone on this planet is the same - but we all carry our own unique set of alleles

Some of these alleles cause or predispose people to various medical conditions such as sickle-cell anemia, cystic fibrosis, breast cancer, Alzheimer’s disease, alcoholism and heart disease

The knowledge of the genes involved in such disorders will make possible the diagnosis and eventually treatment of these genetic disorders

4. The human genome project along with many companion projects have sequenced the complete genomes of many organism on earth and allows us to begin to understand where we fit with all the other life forms found on this planet

Using DNA Technology to Treat Disease

Human insulin, for people suffering from diabetes, was the first human protein made by recombinant DNA technology

Before 1982 the insulin used to treat diabetes was taken from the pancreases of cattle or pigs slaughtered for meat and about 5% of those treated were allergic to the animal derived insulin

Traditional versus Modern Biotechnology

Traditional biotechnology such as selective breeding of desired traits in farm animals or plants is well known but generally takes many generations to accomplish whereas genetic engineering can accomplish this in a single generation

In traditional methods of selective breeding the genetic material recombined is from the same or very closely related species - modern genetic engineering can combine genes from very different organisms

Traditional biotechnologists had no way to manipulate the DNA sequence of genes themselves and genetic engineering can produce new genes never before seen on earth

Should Genetically Modified Organisms (GMOs) be Permitted in Agriculture?

The best transgenic crops have clear advantages to farmers - larger yields

Herbicide-resistant crops allow farmers to rid their fields of weeds by applying the appropriate herbicide that does not effect the crop they are growing while killing all the weeds

Insect resistant crops, decrease the amount pesticides needed and save the farmer money

Scientific Objections to GMOs

1. May be hazardous to human health

2. May be dangerous to the environment

Are GMOs Dangerous to Eat?

Test show that Bt protein is not toxic to mammals and thus not toxic to humans

The Flavr Savr tomato lacked an enzyme that causes tomatoes to get soft as they ripen (won’t bruise during shipment and handling) - they did not taste very good and have disappeared from the grocery store - but they did not make people sick

In 2003 the US Society of Toxicology determined that all currently produced transgenic plants posed no danger to humans

Are GMOs Hazardous to the Environment?

The environmental effects of GMOs is much more debatable

The effect of using Bt crops is the application of less pesticide to fields - thus less pollution in the environment

Are GMOs Hazardous to the Environment?

In eastern Europe where many crops we common use originated, have many weedy relatives in the wild. Thus pollen from a Bt crop could spread the herbicide resistance genes beyond the farmers field and humans could end up with weeds that are resistant to herbicides

It is well known that Bt corn (produces a toxin to kill the European corn borer) is toxic to monarch butterflies larvae when the pollen of the Bt corn lands on the favorite food of monarchs, milkweed plants, and is consumed by the caterpillars

Currently very little of the Bt corn is actually being used & thus the threat is small