DNA Technology
All Materials © Cmassengale

Introduction:

Biotechnology refers to technology used to manipulate DNA

The procedures are often referred to as genetic engineering

DNA is the genetic material of all living organisms

All organisms use the same genetic code

Genes from one kind of organism can be transcribed and translated when put into another kind of organism

For example, human and other genes are routinely put into bacteria in order to synthesize products for medical treatment and commercial use

Human insulin, human growth hormone, and vaccines are produced by bacteria

Recombinant DNA refers to DNA from two different source

Individuals that receive genes from other species are transgenic

Viruses & their Structure:

Viruses contain genetic material but are not living

Host cells are required for their reproduction

Viruses are composed of an inner nucleic acid core (genetic material) and an outer protein coat (capsid)

Viruses that infect animals have an outer envelope (membrane) that is derived from the cell membrane of the host cell may surround the capsid

The genetic material in some viruses is DNA; in others it is RNA

Viral Reproduction:

When viral genetic material enters a cell, it is replicated, transcribed (mRNA is produced) and translated (proteins are produced from the mRNA) by the host cell

By this process, the host cell uses the genetic instructions in the virus to make more viruses

Viral DNA ® mRNA ® protein

If the viral genetic material is RNA, a DNA copy must first be made before transcription and translation can occur

The DNA copy of the viral RNA is called cDNA.

viral RNA ® cDNA ® mRNA ® protein

Bacteriophages:

Bacteriophages are viruses that infect bacteria

Not surrounded by a membrane as the animal-infecting viruses

Virus attaches to the bacteria cell, a viral enzyme digests away a part of the wall, and its viral DNA enters the host cell

Inside the host cell, the viral DNA is transcribed, translated, and replicated

Translation produces protein coats and the enzymes needed in the construction of new virus particles

Viral DNA is replicated

The protein coats and DNA are assembled into new viral particles

The host cell wall to ruptures releasing the newly formed viruses

Upon entering the cell, the viral DNA may instead, become integrated into the bacterial DNA

It is replicated along with the host DNA when the host reproduces

Eventually, it will become transcribed and translated

Retroviruses:

Contain RNA & the enzyme reverse transcriptase

Reverse transcriptase can make a DNA copy of the viral RNA

The new DNA produced from the RNA template is called cDNA

DNA synthesis follows the production of cDNA to produce a double-helix

cDNA then becomes incorporated into the host DNA (called a prophage)

The new viruses escape the host cell by budding

The AIDS virus (HIV) is an example of a retrovirus

Vectors

Vectors are used to transfer genes into a host cell
Plasmids & viruses are the most commonly used vectors
A vector must be capable of self-replicating inside a cell
Viruses are the vectors of choice for animal cells
Marker genes can be used to determine if the gene has been taken up

Plasmids:

Small rings of DNA in bacterial cells
Used to transfer genes to other organisms
Host bacterium takes up the plasmid, which includes the foreign gene
When bacteria reproduce, plasmids with the new gene are also reproduced
This clones (copies) the gene each time the bacteria reproduces

Viruses:

Can accept larger amounts of DNA than plasmids
Once the virus enters the host cell, it also reproduces the foreign gene it carries
The copied gene is "cloned"

Restriction enzymes:

Restriction enzymes were discovered in bacteria

Bacteria use them as a defense mechanism to cut up the DNA of viruses or other bacteria

Hundreds of different restriction enzymes have been isolated

Each restriction enzyme or RE cuts DNA at a specific base sequence

For example, EcoRI always cuts DNA at GAATTC as indicated below

The sequence GAATTC appears three times in the DNA strand below. As a result, the strand is cut into four pieces

Other restriction enzymes cut at different sites, some examples are listed below

Enzyme

Cutting Site

Bam HI GGATCC

Hae III GGCC

Pst I CTGCAG

Hind I GANTC

Sticky Ends & Recombinant DNA:

Fragments of DNA that has been cut with restriction enzymes have unpaired nucleotides at the ends called sticky ends

Sticky ends have complimentary bases, so they could rejoin

If the vector and the gene to be cloned are both cut with the same restriction enzyme, they will both have complimentary sticky ends

After cutting, the 2 DNA samples are mixed

Fragments with complementary sticky ends join together forming recombinant DNA (contains gene from vector & the gene to be cloned)

Enzyme DNA ligase seals the fragments together

Bacteria such as Escherichia coli are capable of taking up DNA from their environment

This process is called transformation

CaCl₂ and a procedure called heat shock are used to make E. coli cells more permeable so that they take up the modified plasmids more readily

Genomic Libraries:

A genome is all of the genes in a particular organism

Bacteria or virus vectors can be used to store fragments of the DNA from another species

The DNA is cut up into fragments, and the different fragments are inserted into bacteria or viruses

The collection of bacteria or viruses is called a genomic library

Polymerase Chain Reaction (PCR):

Used to make many copies of small pieces of DNA

Procedure requires primers, DNA polymerase, and nucleotides

Primers are short chains of about 20 nucleotides that are complimentary to a region in the DNA to be amplified

DNA polymerase cannot continue the process unless it has already been started by primers

Nucleotides are needed because DNA is composed of nucleotide "building blocks"

The DNA is heated to approximately 95^o C to separate the two strands of the double helix

After the strands are separated, the DNA is cooled to about 50^o C, and the primers attach

The temperature is raised to approximately 70^o C so the polymerase will attach to & copy the strand

The DNA replication process repeats itself as the solution is then heated and cooled at regular intervals

DNA Fingerprinting (RFLP Analysis):

In RFLP analysis, the DNA of an organism is cut up into fragments using restriction enzymes producing a large number of short fragments of DNA

Because no two individuals have identical DNA, no two individuals will have the same length fragments

Gel electrophoresis is a technique used to separate the DNA fragments according to their size

The fragments are placed in wells on a sheet of gelatin, and an electric current is applied to the sheet

DNA is negatively charged and will move in an electric field toward the positive pole

The smallest fragments will move the fastest because they are able to move through the pores in the gelatin faster

Bands will be produced on the gelatin where the fragments accumulate

Shortest fragments will accumulate near one end of the gelatin (furthest from the wells), and the longer, slower-moving ones will remain near the other end

DNA bands must be stained to make them visible

Gene Products & Uses of Genetic Engineering:

E. coli is used to produce proteins such as insulin by genetic engineering because it is easily grown

To recover the product, E. coli must be lysed or the gene must be linked to a gene that produces a naturally secreted protein

Yeasts can be genetically engineered and are likely to secrete the gene product continuously

Mammalian cells can be engineered to produce proteins such as hormones for medical use

Plant cells take up a plasmid from Agrobacterium

Plant cells can be engineered and used to produce plants with new properties such as Roundup Ready soybeans

Pseudomonas bacteria has been engineered to produce Bacillus thuringiensis or BT

BT bacteria make a toxin against insects, thus producing a natural insecticide (example - B.T. cotton)

Animal viruses can be engineered to carry a gene for a pathogen's surface protein so the virus can be used as a vaccine

Genetic engineering techniques are being used to map the human genome through the Human Genome Project

Could provide tools for diagnosis and possible repair of genetic disease

Recombinant DNA techniques can be used for genetic fingerprinting

Gene therapy can be used to cure genetic diseases by replacing the defective or missing gene

Bovine growth hormone (BGH) increases milk production in cows by about 10%

Safety and Ethical Issues:

Harmful organisms may be accidentally produced

Organisms that are intended to be released in the environment may be engineered with genes that will eventually kill them

There is little legislation on the use of genetic screening and information produced by screening

The technology is increasing the ability to diagnose genetic diseases pre-natally, adding new complexity to the abortion controversy

Ethical questions have been raised over whether we should modify the genes of humans

Genetic screening and gene therapy are expensive and may be unavailable to the poor

Biological weapons could be created using biotechnology

Enzyme	Cutting Site
Bam HI	GGATCC
Hae III	GGCC
Pst I	CTGCAG
Hind I	GANTC