Scientist examining DNA sequencing autoradiogram on a light box Each group of four strips represents the nucleotide sequence of AGCT (Adenine – Guanine – Cytosine – Thymine) in the DNA of the nematode worm, Onchocerca volvulus, a human parasite responsible for cutaneous onchocerciasis and river blindness in tropical countries.

One of the great achievements of modern science has been the sequencing of the human genome, which was largely completed by 2003. The sequencing of the first complete genome, that of a bacterium, had been achieved in previous years. During the intervening years, researchers accelerated the pace of DNA sequencing, while working on other genomes, aided by the development of faster and faster sequencing machines. These sequencing accomplishments have all depended on advances in DNA technology, starting with the invention of methods for making recombinant DNA. This is DNA in which nucleotide sequences from different sources – often different species – are combined in vitro into the same DNA molecule.

The methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes. Applications of genetic engineering include the manufacture of hundreds of protein products, such as hormones and blood–clotting factors. Using DNA technology, scientists can make recombinant DNA and then introduce it into cultured cells that replicate the DNA and express its genes, yielding a desired protein.

DNA technology has launched a revolution in the area of biotechnology, the manipulation of organisms or their components to make useful products. Practices that go back centuries are forms of biotechnology: for example, the use of microbes to make wine and cheese and the selective breeding of livestock, which exploits naturally occurring mutations and genetic recombination.

Gel electrophoresis Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-used techniques in biochemistry and molecular biology.

As we've understood the concept how the lengthy DNA molecule is fragmented, the next step is to separate the DNA fragments. Here comes the technique named gel electrophoresis. This technique uses a gel as a molecular sieve to separate nucleic acids or proteins on the basis of size, electrical charge, and other physical properties. Because nucleic acid molecules carry negative charges on their phosphate groups, they all travel toward the positive electrode in an electric field. Thus, gel electrophoresis separates a mixture of linear DNA molecules into bands, each consisting of DNA molecules of the same length. The figure here shows the process of how DNA fragments of differing lengths can be separated by agarose gel electrophoresis.

A mixture of DNA fragments is loaded onto a gel, the gel is covered in buffer, and an electric field is generated when the gel box is connected to a power supply (negative electrode on top of the gel, and positive on the bottom). The DNA migrates through the gel toward the positive electrode.

Larger DNA molecules migrate more slowly, and smaller fragments migrate more quickly. Typically a molecular weight marker (MW marker) is included, which is essentially a mixture of DNA fragments of known lengths. The migration of an unknown fragment is compared to the migration of the MW marker and thereby the length of an unknown fragment is determined. The distance of migration is proportional to the length of the DNA fragment. Also, in order to visualize the DNA, the gel must be either cast with, or soaked in, a fluorescent dye. The most common dye used is ethidium bromide, which binds to DNA and fluoresces under UV light.

RFLP in the beta globin gene in sickle cell anemia The wild–type beta–globin gene (red) and the sickle cell anemia beta–globin gene (pink) are shown.

DNA fragments are separated by gel electrophoresis and the bands are transferred to a special membrane that binds single–stranded nucleic acid. Typically the DNA is first denatured in the gel, by soaking it in alkaline buffer, and then it is rapidly neutralized. The gel is laid flat, the membrane is laid over the gel, and a stack of absorbent paper towels is placed on top of the membrane. The absorbent paper will wick the liquid from the gel through the membrane and, as it does so, the DNA will travel from the gel onto the membrane and be bound there. The relative positions of the DNA in the gel are preserved when the DNA is transferred to the membrane. The membrane, also referred to as the blot, is removed and then mixed with a specific probe.

The probe is a single strand of DNA of a specific sequence, typically labeled fluorescently or radioactively. The membrane is placed in a solution with the probe and left for several hours. The probe will anneal (hybridize) to the fragments bound to the membrane that contain complementary DNA sequences. The unbound probe is washed away, and what remains on the blot is the labeled probe hybridized to the fragments that possess complementary DNA. Using this technique, one can detect the presence of a unique fragment of DNA in an entire fragmented genome.

In the adjacent figure shown the procedure of separating DNA molecules. In the figure, this is the part of the gene that is recognized by the probe. The Dde I sites are marked by the stars. The mutation in the beta–globin gene results in the loss of one Dde I site. Genomic DNA is isolated from the cells of normal (wild type) sickle cell carriers (heterozygous for the sickle cell beta–globin gene – one wild–type gene and one sickle cell beta–globin gene) and sickle cell patients (homozygous for the sickle cell beta–globin gene – both genes are sickle cell beta–globin). Remember that cells are diploid and thus have two copies of each gene. The genomic DNA is cleaved with Dde I, the fragments are separated by gel electrophoresis and then transferred to the membrane. The membrane is hybridized to labeled beta–globin DNA. The wild–type gene has two fragments and the sickle cell gene has only one larger fragment.

Steps involved in cloning a human gene into a bacterial plasmid. In this simplified diagram of gene cloning in the laboratory, we start with a plasmid isolated from a bacterial cell and a gene of interest from another organism. Only one copy of the plasmid and one copy of the gene of interest are shown at the top of the figure, but the starting materials would include many copies of each

Using one or more somatic cells from a multicellular organism to make another genetically identical individual is called cloning, and is common in plants. Cloning has been tried in mammals, including mice, cats, cows, horses and pigs. Environmental influences and random phenomena can play a significant role during development. However, only a small percentage of cloned embryos develop normally to birth. And like Dolly, the first cloned lamb, many cloned animals exhibit various defects. Cloned mice, for instance, are prone to obesity, pneumonia, liver failure, and premature death.

Scientists believe that even cloned animals that appear normal are likely to have subtle defects. In the nuclei of fully differentiated cells, a small subset of genes is turned on and expression of the rest is repressed. This regulation often is the result of epigenetic changes in chromatin, such as acetylation of histones or methylation of DNA. Many of these changes 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.

Researchers have found that the DNA in embryonic cells from cloned embryos, like that of differentiated cells, often has more methyl groups than does the DNA in equivalent cells from uncloned embryos of the same species. This finding suggests that the reprogramming of donor nuclei is not always complete. Because DNA methylation helps regulate gene expression, misplaced methyl groups in the DNA of donor nuclei may interfere with the pattern of gene expression necessary for normal embryonic development.

The cloning of genes through this method has been supplemented by amplification of specific DNA sequences using the polymerase chain reaction. Due to the availability of the complete genomic sequence of many organisms, it is relatively easy to design PCR primers specific for the amplification of any portion of the genome. Gene cloning and PCR allow scientists to make a large amount of DNA from only a small fragment. Thus, DNA cloning is the method for amplifying the gene of interest in in vivo conditions, whereas the more advanced PCR technique happens in in vitro conditions, and where the amplification of DNA is rapid. How do these technologies work?

DNA replication. Technician with rows of thermocyclers used in research into the human genome at Celera Genomics, Maryland, USA. Thermocyclers clone DNA using a polymerase chain reaction (PCR). Celera aimed to sequence every gene in human DNA (deoxyribonucleic acid) by 2001. Human DNA is made up of some 30,000 genes among over 3 billion base pairs. Celera sequences DNA fragments and uses supercomputers to put the pieces together. This is a cruder but faster method than that used by the publicly–funded Human Genome Project. The complete human genome map will allow improved drug design and a greater understanding of genetic diseases.

PCR is only feasible because of the discovery and isolation of thermostable DNA polymerase. The first thermostable DNA polymerase was isolated from the bacterium Thermus aquaticus, which normally grows in the water of hot springs. This enzyme, called Taq DNA polymerase, is optimally active at 72°C and can retain its activity after successive heating to denature the template DNA. With each successive cycle of DNA synthesis, the number of copies of the target sequence is doubled. Typically 20–30 cycles of DNA synthesis will be performed, resulting in 106–109 copies of the target sequence.

If we go into details of PCR mechanism, the procedure is as follows:

Step 1: At the start of PCR, the DNA from which a segment is to be amplified, an excess of the two primer molecules, the four deoxyriboside triphosphates and the DNA polymerase are mixed together in the reaction mixture. The following operations are now performed sequentially.

Step 2: The mixture is now cooled to a temperature (generally 40–60 degree C) that permits annealing of the primer to the complementary sequences in the DNA; these sequences are located at the 3' ends of the two strands of the desired segment. This step is called annealing.

Step 3: The temperature is now so adjusted that the DNA polymerase synthesizes the complementary strands by utilizing 3' – OH of the primers; this reaction is same as that occurs in vivo during replication of the leading strand of a DNA duplex. The primers are extended towards each other so that the DNA segment lying between the two primers is copied; this is ensured by employing primers complementary to the 3' ends of the segment to be amplified. In case of Taq polymerase, the optimum temperature for synthesis is between 70-75 degree C; the temperature of reaction mixture is, therefore, adjusted to this temperature. This situation has the following additional advantages. Between 70–75 degree C, the base pairing between about 20 bases long primers and the DNA is much more specific than at 37 degree C, the optimal temperature for E.coli DNA polymerase. This minimizes the chances of annealing of primers to imperfectly matched sequences and thereby, amplification of unwanted DNA. The specificity of annealing is further increased by selecting appropriate conditions like ionic strength, primer length etc. The completion of step 3 completes the first cycle of amplification; each cycle may take few (ordinarily 1–3) minutes.

Step 4: The next cycle of amplification is initiated by denaturation (step 1), which separates the newly synthesized DNA strands from the old DNA strands.

Step 5: Annealing allows the primers to base pair with both the new and old strands, the total number of strands being twice their original number.

Step 6: Synthesis of new strands takes place, which doubles the number of copies of the desired DNA segment present at the end of step 1. This completes the second cycle.

Research on the human genome Dr Peter Lichter, of Yale Medical School, using a light microscope to do fine mapping of long DNA fragments on human chromosomes using a technique known as non–radioactive in–situ hybridisation. The chromsomes appear in red on the monitor screen, whilst the DNA fragments (called probes) appear yellow/green. Mapping chromosomes may be regarded as a physical survey of each chromosome to find the location of genes or other markers. Mapping & sequencing are the two main phases of the genome project; an ambitious plan to build a complete blueprint of human genetic information.

A genome map may be defined as a detailed schematic description of the structural and functional organization of all the chromosomes in the genome of an organism. The most ambitious mapping project to date has been sequencing of the human genome, officially begun as the Human Genome Project.

Human Genome Project The Human Genome Project was organized to map and to sequence the human genome. In May 2007, the New York Times announced that the full genome of DNA pioneer James D. Watson had been recorded. The article noted that some scientists believe this to be the gateway to upcoming personalized genomic medicine. Many genomes have been sequenced by various genome projects. The estimated number of genes in the human genome has been repeatedly revised downward since the completion of the Human Genome Project; current estimates place the human genome at just under 3 billion base pairs and about 20,000 – 25,000 genes.

A recent Science article gives a number of 20,488 protein – coding genes, with perhaps 100 more yet to be discovered. Genomics can also be considered a subfield of bioinformatics, which uses computational approaches to analyze large sets of biological data. Bioinformatics involves the manipulation, searching and data mining of DNA sequence data.

String searching or matching algorithms, which find an occurrence of a sequence of letters inside a larger sequence of letters, were developed to search for specific sequences of nucleotides. The genome of an organism is its whole hereditary information and is encoded in the DNA (or for some viruses, RNA). This includes both the genes and the non–coding sequences of the DNA. The total complement of genes in an organism or cell is known as its genome. Genomic DNA is located in the cell nucleus of eukaryotes, as well as small amounts in mitochondria and chloroplasts. More precisely, the genome of an organism is a complete genetic sequence on one set of chromosomes; for example, one of the two sets that a diploid individual carries in every somatic cell. The term genome can also be applied to that stored within organelles that contain their own DNA, as with the mitochondrial genome or the chloroplast genome.

Three – stage approach to map an entire genome. Starting with a cytogenetic map of each chromosome, researchers with the Human Genome Project proceeded through three stages of mapping to reach the ultimate goal, the nearly complete nucleotide sequence of every chromosome.

An analogy to the human genome stored on DNA is that of instructions stored in a book:

• The book over one billion words long.
• The book is bound 5000 volumes, each 300 pages long.
• The book fits into a cell nucleus the size of a pinpoint.

A copy of the book (all 5000 volumes) is contained in every cell (except red blood cells) as a strand of DNA over two meters in length. In many species, only a small fraction of the total sequence of the genome encodes protein.

For example, only about 1.5% of the human genome consists of protein–coding exons, with over 50% of human DNA consisting of non–coding repetitive sequences. The reasons for the presence of so much non–coding DNA in eukaryotic genomes and the extraordinary differences in genome size, or C–value, among species represent a long–standing puzzle known as the "C–value enigma".

DNA sequences that do not code protein may still encode functional non–coding RNA molecules, which are involved in the regulation of gene expression. Some non–coding DNA sequences play structural roles in chromosomes. Telomeres and centromeres typically contain few genes, but are important for the function and stability of chromosomes. An abundant form of non–coding DNA in humans are pseudogenes, which are copies of genes that have been disabled by mutation.

These sequences are usually just molecular fossils, although they can occasionally serve as raw genetic material for the creation of new genes through the process of gene duplication and divergence. In addition to mapping human DNA, researchers with the Human Genome Project are also working on the genomes of other species important in biological research. They have completed sequences for E. coli and numerous other prokaryotes, Saccharomyces cerevisiae (yeast), Caenorhabditis elegans (nematode), Drosophilia melanogaster (fruit fly), Mus musculus (mouse), and quite a few others.

Whole–genome shotgun approach to sequencing. In this approach, developed by Celera Genomics, random DNA fragments are sequenced and then ordered relative to each other. Compare this approach with the hierarchical, three–stage approach shown in the figure.

It is also known as full genome sequencing, complete genome sequencing, or entire genome sequencing. It is a laboratory process that determines the complete DNA sequence of an organism's genome at a single time. This entails sequencing all of an organism's chromosomal DNA as well as DNA contained in the mitochondria and, for plants, in the chloroplast.

Almost any biological sample—even a very small amount of DNA or ancient DNA—can provide the genetic material necessary for full genome sequencing. Such samples may include saliva, epithelial cells, bone marrow, hair (as long as the hair contains a hair follicle), seeds, plant leaves, or anything else that has DNA–containing cells.

Because the sequence data that is produced can be quite large (for example, there are approximately six billion base pairs in each human diploid genome), genomic data is stored electronically and requires a large amount of computing power and storage capacity.

Full genome sequencing would have been nearly impossible before the advent of the microprocessor, computers. The three stages of mapping (genetic mapping, physical mapping and DNA sequencing) explained above overlap in a way that our simplified version does not portray, but they accurately represent the overarching strategy employed in the Human genome project. In 1992, emboldened by advances in sequencing and computer technology, molecular biologist J. Craig Venter devised an alternative approach to the sequencing of whole genomes. His idea was essentially to skip the genetic mapping and physical mapping stages and start directly with the sequencing of random DNA fragments. Powerful computer programs would then assemble the resulting very large number of overlapping short sequences into a single continuous sequence.

Proteomics, the systematic study of full protein sets Example: Antibody response, conceptual image. Computer artwork representing immunoglobulin (Ig) molecules (red and yellow) responding to a pathogen. Each Y–shaped molecule has two arms (top) that can bind to specific antigens, for instance bacterial or viral proteins. In doing this they mark the antigen for destruction by phagocytes, white blood cells that ingest and destroy foreign bodies. Antibodies can also kill some pathogens directly, and can neutralize toxins.
Genomics and proteomics are enabling biologists to now be in a position to complete catalogs of genes and proteins – a listing of all the "parts" that contribute to the operation of cells, tissues, and organisms.

The next step after mapping and sequencing genomes is Proteomics, the systematic study of full protein sets (proteomes) encoded by genomes. One challenge is the sheer number of proteins in humans and the proteins are extremely varied in structure, chemical and physical properties. Because proteins are the molecules that actually carry out cell activities, we must study them to learn how cells and organisms function. Genomics and proteomics are enabling biologists to approach the study of life from an increasingly global perspective. Biologists are now in a position to complete catalogs of genes and proteins – a listing of all the "parts" that contribute to the operation of cells, tissues, and organisms.

Bioinformatics as a discipline arose out of the need to introduce order into the massive data sets produced by the new technologies of molecular biology: large–scale DNA sequencing, measurements of RNA concentrations in multiple gene expression arrays, and new profiling techniques in proteomics. As such, bioinformatics integrates a number of traditional quantitative sciences such as mathematics, statistics, computer science and cybernetics with biological sciences such as genetics, genomics, proteomics and molecular evolution. Another exciting prospect is our increasing understanding of the spectrum of genetic variation in humans. In other words, if you could compare your personal DNA sequence with that of a person of the same gender, either sitting next to you or on the other side of the world, you would find them to be 99.9% identical.

Most of our diversity seems to be in the form of single nucleotide polymorphisms (SNPs, pronounced as "snips"), which are single base–pair variations in the genome, usually detected by sequencing. In human genome, SNPs occur on average about once in 1,000 base pairs. This will be useful as genetic markers for studying human evolution, the differences between human populations, and the migratory routes of human populations throughout history. SNPs and other polymorphisms in non-coding and coding DNA will also be valuable markers for identifying disease genes and genes that affect our health in more subtle ways.

DNA fingerprints from a murder case This autoradiograph shows that DNA in blood from the defendant's clothes matches the DNA fingerprint of the victim but differs from the DNA fingerprint of the defendant. This is evidence that the blood on the defendant's clothes came from the victim, not the defendant. The three DNA samples were subjected to Southern blotting using radioactive probes. The DNA bands resulting from electrophoresis were exposed to probes for several different RFLP markers in succession, with the previous probe washed off before the next one was applied.

DNA is present in nearly every cell of our body, and we leave cells behind everywhere we go without even realizing it. Flakes of skin, drops of blood, hair, and saliva all contain DNA that can be used to identify us. In fact, the study of forensics, commonly used by police departments and prosecutors around the world, frequently relies upon these small bits of shed DNA to link criminals to the crimes they commit. This fascinating science is often portrayed on popular television shows as a simple, exact, and infallible method of finding a perpetrator and bringing him or her to justice.

In truth, however, teasing out a DNA fingerprint and determining the likelihood of a match between a suspect and a crime scene is a complicated process that relies upon probability to a greater extent than most people realize. Government–administered DNA databases, such as the Combined DNA Index System (CODIS), do help speed the process, but they also bring to light complex ethical issues involving the rights of victims and suspects alike. Thus, understanding the ways in which DNA evidence is obtained and analyzed, what this evidence can tell investigators, and how this evidence is used within the legal system is critical to appreciating the true ethical and legal impact of forensic genetics.

To perform DNA fingerprinting, DNA is obtained from an individual's blood cells, hair fibers, skin fragments, or other tissue. The DNA is then extracted from the cells and digested with enzymes. The resulting fragments are separated by a process called electrophoresis. Electrophoresis is a process in which electrical charges separate DNA fragments according to size. The separated DNA fragments are then detected with DNA probes and used to develop a fingerprint. A statistical evaluation enables the forensic pathologist to compare a suspect's DNA with the DNA recovered at a crime scene and to state with a high degree of certainty (usually 99 percent) that the suspect was at the crime scene.

Production of transgenic animals. A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. The foreign gene is constructed using recombinant DNA methodology and the ultimate goal is to produce animals with incredible features.

DNA technology is now routinely used to make vaccines and growth hormones for treating farm animals. Scientists can also introduce a gene from one animal into the genome of another animal, which is then called a transgenic animal. The mechanism for creating one can be described in three steps:

• Healthy egg cells are removed from a female of the host animal and fertilized in the laboratory.
• The desired gene from another species is identified, isolated, and cloned.
• The cloned genes are injected directly into the eggs, which are then surgically implanted in the host female, where the embryo undergoes a normal development process.

It is hoped that this process will provide a cheap and rapid means of generating desired enzymes, other proteins, and increased production of meat, wool, and other animal products through common, natural functions. Ever since 1997 when Dolly was cloned, research and experimentation to clone useful livestock has continued unceasingly. The attractiveness of cloning is the knowledge that the offspring will be genetically identical to the parent as in asexual reproduction. Four steps describe the general process:

• A differentiated cell, one that has become specialized during development, with its diploid nucleus is removed from an animal to provide the DNA source for the clone.
• An egg cell from a similar animal is recovered and the nucleus is removed, leaving only the cytoplasm and cytoplasm organelles.
• The two egg cells are fused with an electric current to form a single diploid cell, which then begins normal cell division.
• The developing embryo is placed in a surrogate mother, who then undergoes a normal pregnancy.

A laboratory chemostat containing a culture of Escherichia coli, genetically modified to produce the bioplastic, polyhydroxybutyrate (PHB) PHB is a non-toxic biodegradable plastic suitable for use as food packaging, plastic cutlery and drinking vessels. E. coli is a fast growing bacterium suited to bulk liquid culture. It does not naturally make PHB. Genetic modification is used to control the amount and type of PHB produced by E. coli in culture. The picture shows a research-scale chemostat containing E. coli into which genes for PHB synthesis inserted. At and above the liquid culture surface, the plastic can be seen adhering to the inside of the chemostat (off-white strands, center and to left). PHB is expensive compared to oil–based plastics. Current research concerns the possible use of waste water as a feedstock in order to increase the competitiveness of the product.

Despite the fact that the genes being transferred occur naturally in other species, there are unknown consequences to altering the natural state of an organism through foreign gene expression. After all, such alterations can change the organism's metabolism, growth rate, and/or response to external environmental factors. These consequences influence not only the GMO itself, but also the natural environment in which that organism is allowed to proliferate. Potential health risks to humans include the possibility of exposure to new allergens in genetically modified foods, as well as the transfer of antibiotic–resistant genes to gut flora.

Horizontal gene transfer of pesticide, herbicide, or antibiotic resistance to other organisms would not only put humans at risk, but it would also cause ecological imbalances, allowing previously innocuous plants to grow uncontrolled, thus promoting the spread of disease among both plants and animals.

GMOs benefit mankind when used for purposes such as increasing the availability and quality of food and medical care, and contributing to a cleaner environment. If used wisely, they could result in an improved economy without doing more harm than good, and they could also make the most of their potential to alleviate hunger and disease worldwide. However, the full potential of GMOs cannot be realized without due diligence and thorough attention to the risks associated with each new GMO on a case–by–case basis.

Finally, ethical considerations, as well as concern about potential environmental and health hazards, will likely slow some applications of biotechnology. There is always danger that too much regulation will stifle basic research and its potential benefits. However, the power of DNA technology and genetic engineering – our ability to profoundly and rapidly alter species that have been evolving for millennia – demands that we proceed with humility and caution.