DNA Tools and Biotechnology

The DNA Toolbox

Recently, the genome sequences of two extinct species—Neanderthals and wooly mammoths—have been completed.
Advances in sequencing techniques make genome sequencing increasingly faster and less expensive.

Biotechnology

Biotechnology is the manipulation of organisms or their components to make useful products.
The applications of DNA technology affect everything from agriculture to criminal law to medical research.

DNA Sequencing and DNA Cloning as Tools

The complementarity of the two DNA strands is the basis for nucleic acid hybridization, the base pairing of one strand of nucleic acid to the complementary sequence on another strand.
Genetic engineering is the direct manipulation of genes for practical purposes.

DNA Sequencing

Researchers can exploit the principle of complementary base pairing to determine a gene’s complete nucleotide sequence, called DNA sequencing.
The first automated procedure was based on a technique called dideoxy or chain termination sequencing, developed by Sanger.
“Next-generation sequencing” techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments.
Thousands or hundreds of thousands of fragments (400–1,000 nucleotides long) are sequenced in parallel.
This is a type of “high-throughput” technology.
In “third-generation sequencing,” the techniques used are even faster and less expensive than the previous.

Making Multiple Copies of a Gene or Other DNA Segment

To work directly with specific genes, scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning.
Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome.
Researchers can insert DNA into plasmids to produce recombinant DNA, a molecule with DNA from two different sources.
Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA.
This results in the production of multiple copies of a single gene.
The production of multiple copies of a single gene is a type of DNA cloning called gene cloning.
A plasmid used to clone a foreign gene is called a cloning vector.
Bacterial plasmids are widely used as cloning vectors because they are readily obtained, easily manipulated, easily introduced into bacterial cells, and once in the bacteria they multiply rapidly.
Gene cloning is useful for amplifying genes to produce a protein product for research, medical, or other purposes.

Using Restriction Enzymes to Make a Recombinant DNA Plasmid

Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites.
A restriction enzyme usually makes many cuts, yielding restriction fragments.
The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends”.
Sticky ends can bond with complementary sticky ends of other fragments.
DNA ligase is an enzyme that seals the bonds between restriction fragments.
To check the recombinant plasmid, researchers might cut the products again using the same restriction enzyme.
To separate and visualize the fragments produced, gel electrophoresis would be carried out.
This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size, charge, or other physical properties.

Amplifying DNA: The Polymerase Chain Reaction (PCR)

The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA.
A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules.
The key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase.
PCR uses a pair of primers specific for the sequence to be amplified.
PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells.
PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors.
The resulting clones are sequenced and error-free inserts selected.

Expressing Cloned Eukaryotic Genes

After a gene has been cloned, its protein product can be produced in larger amounts for research.
Cloned genes can be expressed as protein in either bacterial or eukaryotic cells.

Bacterial Expression Systems

Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells.
To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector, a cloning vector that contains a highly active bacterial promoter.
Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes.
Researchers can avoid this problem by using cDNA, complementary to the mRNA, which contains only exons.

Eukaryotic DNA Cloning and Expression Systems

Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells, such as yeasts, as hosts for cloning and expressing genes.
Even yeasts may not possess the proteins required to modify expressed mammalian proteins properly.
In such cases, cultured mammalian or insect cells may be used to express and study proteins.
One method of introducing recombinant DNA into eukaryotic cells is electroporation, applying a brief electrical pulse to create temporary holes in plasma membranes.
Alternatively, scientists can inject DNA into cells using microscopically thin needles.
Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination.

Cross-Species Gene Expression and Evolutionary Ancestry

The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species.
For example, Pax-6 is a gene that directs the formation of a vertebrate eye; the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye).
The Pax-6 genes in flies and vertebrates can substitute for each other.

Studying Gene Expression and Function

Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function.

Analyzing Gene Expression

The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made.

Studying the Expression of Single Genes

mRNA can be detected by nucleic acid hybridization with complementary molecules.
These complementary molecules, of either DNA or RNA, are nucleic acid probes.
In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism.
Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time.
Reverse transcriptase is added to mRNA to make complementary DNA (cDNA), which serves as a template for PCR amplification of the gene of interest.
The products are run on a gel and the mRNA of interest is identified.

Studying the Expression of Interacting Groups of Genes

Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays.
DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions.
With rapid and inexpensive sequencing methods available, researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them.
By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to the understanding of disease and suggest new diagnostic targets.

Determining Gene Function

One way to determine function is to disable the gene and observe the consequences.
Using in vitro mutagenesis, mutations are introduced into a cloned gene, altering or destroying its function.
When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype.
Gene expression can also be silenced using RNA interference (RNAi).
Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the gene’s mRNA.
In humans, researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition.
These genome-wide association studies test for genetic markers, sequences that vary among individuals.
SNPs (single nucleotide polymorphisms), single nucleotide variants, are among the most useful genetic markers.
SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene.
SNPs are rarely directly involved in the disease; they are most often in noncoding regions of the genome.

Cloned Organisms and Stem Cells

Organismal cloning produces one or more organisms genetically identical to the “parent” that donated the single cell.
A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely, or under certain conditions can differentiate into one or more types of specialized cells.

Cloning Plants: Single-Cell Cultures

In plants, cells can dedifferentiate and then give rise to all the specialized cell types of the organism.
A totipotent cell, such as this, is one that can generate a complete new organism.
Plant cloning is used extensively in agriculture.

Cloning Animals: Nuclear Transplantation

In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell.
Experiments with frog embryos have shown that a transplanted nucleus can often support normal development of the egg.
However, the older the donor nucleus, the lower the percentage of normally developing tadpoles.

Reproductive Cloning of Mammals

In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell.
Dolly’s premature death in 2003, as well as her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus.
Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs.
CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent”.
Cloned animals do not always look or behave exactly the same.

Faulty Gene Regulation in Cloned Animals

In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth, and many cloned animals exhibit defects.
Many epigenetic changes, such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development.

Stem Cells of Animals

Stem cells are relatively unspecialized cells that can both reproduce indefinitely and, under certain conditions, differentiate into one or more specialized cell types.

Embryonic and Adult Stem Cells

Many early embryos contain stem cells capable of giving rise to differentiated embryonic cells of any type.
In culture, these embryonic stem cells reproduce indefinitely.
Depending on culture conditions, they can be made to differentiate into a variety of specialized cells.
Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed.
Embryonic stem (ES) cells are pluripotent, capable of differentiating into many different cell types.
The ultimate aim of research with stem cells is to supply cells for the repair of damaged or diseased organs.
ES cells present ethical and political issues.

Induced Pluripotent Stem (iPS) Cells

Researchers can treat differentiated cells, and reprogram them to act like ES cells.
Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells.
iPS cells can perform most of the functions of ES cells.
iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients.

Practical Applications of DNA-Based Biotechnology

Many fields benefit from DNA technology and genetic engineering.

Medical Applications

One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases.
Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases.
The genes and their products are then potential targets for prevention or therapy.

Diagnosis and Treatment of Diseases

Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers, then sequencing the amplified product to look for the disease-causing mutation.
SNPs may be associated with a disease-causing mutation.
SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer.

Human Gene Therapy

Gene therapy is the alteration of an afflicted individual’s genes.
Gene therapy holds great potential for treating disorders traceable to a single defective gene.
Vectors are used for delivery of genes into specific types of cells, for example bone marrow.
Gene therapy provokes both technical and ethical questions.

Pharmaceutical Products

Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases.
The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor.
This approach is feasible for treatment of cancers in which the molecular basis is well-understood.

Protein Production in Cell Cultures

Host cells in culture can be engineered to secrete a protein as it is made, simplifying the task of purifying it.
This is useful for the production of insulin, human growth hormones, and vaccines.

Protein Production by “Pharm” Animals

Transgenic animals are made by introducing genes from one species into the genome of another animal.
Transgenic animals are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use.

Forensic Evidence and Genetic Profiles

An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids.
DNA testing can identify individuals with a high degree of certainty.
Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs).
STRs are variations in the number of repeats of specific DNA sequences.
PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths.
The probability that two people who are not identical twins have the same STR markers is exceptionally small.
As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence.

Environmental Cleanup

Genetic engineering can be used to modify the metabolism of microorganisms.
Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials.

Agricultural Applications

DNA technology is being used to improve agricultural productivity and food quality.
Genetic engineering of transgenic animals speeds up the selective breeding process.
Beneficial genes can be transferred between varieties or species.
Agricultural scientists have endowed a number of crop plants with genes for desirable traits.
The Ti plasmid is the most commonly used vector for introducing new genes into plant cells.
Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance, increased resistance to pests, increased resistance to salinity, and improved nutritional value of crops.

Safety and Ethical Questions Raised by DNA Technology

Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures.
Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology.
Most public concern about possible hazards centers on genetically modified (GM) organisms used as food.
Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives.
Other worries include the possibility that transgenic protein products might cause allergic reactions.
As biotechnology continues to change, so does its use in agriculture, industry, and medicine.
National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology.