For the microarray image shown on this slide, let us assume that each spot on the array contains the DNA of a different form of a polymorphic marker. For every form or variation of the marker, there is a separate spot on the array. DNA from the test specimen is applied to the array and allowed to hybridize (bind specifically) to the DNA on the microarray. This hybridization is based on the complementarity of the DNA double helix. Under the proper conditions, the DNA from the specimen will find its complementary counterpart on the array and hybridize (bind) to it. The DNA from the specimen is labeled with a fluorescent tag so that it can be seen with a color camera. In this way, the specimen can be genotyped for each marker represented on the array, based on the pattern of spots to which it hybridizes. For example, if a dimorphic marker (marker with two forms– A and B) is used, test samples can be genotyped as AA, BB or AB based on the pattern of fluorescence on the array. Using this technology, the entire genome of a specimen can be investigated for a large number of markers simultaneously, significantly reducing the labor, time and cost involved in the analysis. Imagine that for a particular marker a preponderance of the B allele is found in the case group but not in the control group: one might suspect that there is a susceptibility gene somewhere in the region of that particular marker. The genomic region surrounding the marker could then be further investigated to attempt to identify disease-associated genes and mutations.

Microarrays also can be used for other purposes as well, such as investigating gene expression in cells. In this application of microarray technology, DNA sequences representing all (or many) of the genes in the genome are immobilized on the array. Fluorescently labeled probes derived from the RNA of a particular cell type are applied to the array to see which genes are being expressed in the cells (and which are not). The spots corresponding to expressed genes will light up because a fluorescently labeled specimen hybridizes there. Spots corresponding to unexpressed genes will remain unlabelled. Under the proper experimental conditions, quantitation of the amount of gene expression also may be possible. This is a useful approach for comparing gene expression between normal and disease tissue. Any differences found could indicate candidate genes associated with susceptibility to or progression of the disease.

To interpret the microarray shown in this slide in a slightly different way, let us now assume that this is a gene expression microarray and that there is a spot on the microarray bearing DNA from every gene in the human genome. Let us also assume that control cDNA, cDNA derived from normal tissue has been tagged with GREEN fluorescent dye and hybridized to the target DNA on the microarray, and that sample cDNA, cDNA derived from diseased tissue is tagged with RED fluorescent dye and hybridized to the target DNA on the microarray. The spots that show green suggest that there is no expression of that particular gene in the disease tissue. The spots that show red suggest that there is no expression of that particular gene in normal tissue. The spots that show yellow would suggest that there is expression of that particular gene in both normal and disease tissue. The spots that show no fluorescence suggest that there is no expression of that particular gene in either normal or disease tissue. Discovery of the differences in gene expression between normal and disease tissue allows researchers to focus their studies on the genes whose expression differs between the normal and disease state and accelerate their understanding of the biochemical pathways that might be activated, or inactivated, in association with a particular disease.

Another use of microarray technology is to search for small gains or losses in genomic DNA in patients with particular disorders. For this application, DNA representing a large percentage of the DNA in the human genome is ordered on an array. A specimen from a patient with a disorder of unknown etiology is labeled with one fluorescent dye and mixed with equal quantities of control DNA from a person without the disorder that has been labeled with a different fluorescent dye. These two DNA samples are then allowed to hybridize to the DNA on the array. By analyzing the fluorescence bound to each of the spots on the array, scientists can determine if portions of the genome are missing from the patient DNA, or occur in more copies than usual in the patient DNA. In this way, researchers can identify regions of the genome where losses or gains of genetic information might be associated with the symptoms in the patient. This is called comparative genomic hybridization.

Restriction enzymes are enzymes produced by bacteria that recognize specific DNA sequences and cut the DNA strand at those sequences. Restriction enzymes can be used to cut DNA for a variety of purposes.

Restriction enzymes can be used to digest the genomic (chromosomal) DNA of an organism and generate a set of DNA fragments of manageable size. For example, digested DNA can be run on electrophoresis gels for evaluation of banding patterns. Variations in the banding patterns (pattern of restriction digestion) of certain genes between individuals affected by a genetic disease and those not affected may indicate the presence of an underlying molecular genetic alteration associated with the disease. Restriction analysis can also be used to detect polymorphic sites that, while not associated with disease, still interrupt the ability of a restriction enzyme to cut a particular segment of DNA. Restriction enzyme digestion of polymorphic sites and evaluation of the banding patterns that resulted were the basis for early DNA fingerprinting methods. Today, it is more common to find forensic DNA technology using length variations in repetitive elements to identify and distinguish among individuals because repetitive sequences can be more polymorphic (have more forms) than restriction fragments (gain or loss of restriction digestion) and have greater power to distinguish among individuals.

In the creation of DNA libraries, the DNA of an organism is digested (cut into fragments) with a restriction enzyme and cloned into vectors (bacteriophage, plasmid, cosmid) for further evaluation in the laboratory. Vectors are small, independently replicating DNA molecules carried by viruses or bacteria that can be manipulated in the laboratory to carry, and copy, genes of interest to researchers. Libraries can also be constructed using RNA as the starting material to synthesize complementary DNA molecules. In this case, a library called a cDNA library is created. Because cDNA libraries are based on RNA (transcribed genes) rather than genomic (total cellular) DNA, they are enriched for expressed (transcribed) genes whereas genomic DNA libraries contain all (or most of) the genes from an organism regardless of whether they were expressed (transcribed). Separate cDNA libraries from various individual cell types can be investigated to discover differences in gene expression between different types of tissues.

Genes isolated from genomic DNA by restriction digestion can be cloned into vectors (bacteriophage, plasmid, cosmid) for further evaluation in the laboratory. Vectors are small, independently replicating DNA molecules carried by viruses or bacteria that can be manipulated in the laboratory to  carry, and copy, genes of interest to researchers. In the cloning process, target DNA is cut by a restriction enzyme. The vector DNA is cut by the same enzyme. The target and vector DNA are mixed together and DNA ligase is added, to join the ends of the target DNA to the ends of the vector DNA. After ligation, the vector is once again a circle but now it carries a piece of the target DNA inserted into the cloning site. The recombinant vector is then transferred into cells in a process called transfection. Once inside the cells, the vector and its cloned gene can be cultured to create millions of copies of the target DNA. Plasmids that replicate in bacterial cells are an efficient mechanism by which genes can be replicated in vitro, but they are limited in that the target DNA cloned into the cloning site can only be of a certain size. Fragments too large are not able to be propagated by the bacterial cells efficiently. Other vectors such as cosmids and phages that replicate in either bacteria or in eukaryotic cells can accommodate larger pieces of cloned DNA.

Genes isolated from genomic DNA by restriction digestion can be cloned into vectors for further analysis. Cloning vectors come in a number of varieties, including phage, plasmid, cosmid, and others. This slide shows a map of the bacterial plasmid vector called pUC19. On the right is the multiple cloning site (purple box labeled MCS). This site contains a number of different restriction enzyme digestion sites, allowing DNA cut by a variety of enzymes to be cloned into this vector. Once inside the bacterial cells, the plasmid and its cloned gene can be cultured to create millions of copies of the target DNA. Plasmids are efficient ways to replicate genes in vitro, but they are limited in that the target DNA cloned into the MCS can only be of a certain size. Fragments too large are not able to be propagated by the bacterial cells efficiently. Other vectors, such as cosmids and phages, that replicate in either bacterial or eukaryotic cells can accommodate larger pieces of cloned DNA.

On the left side of the plasmid drawing is an ampicillin resistance gene (ampR). This gene allows any bacteria harboring this plasmid to survive treatment with the antibiotic ampicillin. In this way, cells that have taken up the recircularized plasmid, called transfected, survive in cultures containing ampicillin, while those that do not carry the plasmid die. Therefore, only bacteria carrying the plasmid are grown, enriching the culture for the cloned target DNA. Naturally occurring plasmids, not genetically engineered ones, are the basis for much of the antibiotic resistance seen in bacterial diseases today.

Southern analysis permits the detection of mutations and polymorphisms in genes because changes in the sequence of genes result in changes in restriction patterns. In Southern analysis, genomic DNA is isolated from a patient’s cells and digested with an enzyme. The resulting DNA fragments are run on an agarose gel for separation according to size. To stabilize the DNA in its electrophoretic separation pattern, the DNA is hybridized (affixed) to a nylon membrane. A labeled DNA probe (short sequence of DNA tagged isotopically or chemically) specific for the gene of interest is mixed with the membrane under conditions that allow the probe to find and bind to (hybridize with) its complementary DNA on the membrane, resulting in a characteristic banding pattern for the normal gene upon isotopic or chemical signal detection. This approach can be used to analyze a particular nucleotide change if the change occurs at the site of action by a restriction enzyme and alters the ability of the enzyme to cut the DNA. This approach also can detect alterations in genes that disrupt the regular banding pattern because of deletions or other rearrangements in the gene that span restriction sites. Southern analysis also can be effective in identifying changes in the size of repetitive elements, such as triplet repeats that are associated with disease. In many cases, the changes in size of the gene associated with expansion of the repeat sequence is large enough to be seen on Southern analysis as a change in the size of the band associated with the gene. In cases of smaller repeat expansions a different type of gel substance, called polyacrylamide, is used because it is better able to separate smaller sized DNA fragments.

In the image on the slide, genomic DNA is digested with a restriction enzyme and separated by gel electrophoresis according to fragment size. The DNA is then transferred from the gel to a nylon membrane and hybridized with a radioactively labeled probe unique to the sequence of interest. In this case, the probe is complementary to the beta-globin gene and is designed to detect the mutation associated with sickle cell disease, which interrupts a restriction enzyme site. The sickle cell-associated genes remain undigested by the enzyme while the normal gene sequence is cut by the enzyme into a smaller piece of DNA. In the lane labeled A, an individual with two normal copies of the beta-globin gene was analyzed. The size of the fragment seen on the autoradiograph is that of the digested DNA, indicating two normal alleles and no sickle cell-associated allele. In the column labeled S, an individual with sickle cell disease and two copies of the sickle cell gene was analyzed. The size of the fragment seen is larger than the fragment in column A, indicating that the restriction enzyme failed to cut the DNA due to the presence of two copies of the sickle cell-associated mutation. In the column labeled AS, an individual with one copy of the normal gene and one copy of the sickle cell-associated gene was analyzed. Two bands are seen: one for the digested smaller allele on the bottom; and, one for the uncut sickle cell-associated allele on the top. This individual is a carrier for sickle cell disease.

The haploid human genome (23 chromosomes) contains ~3 billion base pairs of DNA. The diploid human genome (23 pairs of chromosomes) contains ~6 billion base pairs of DNA.

Only 1.5-3% of the human genome codes for proteins. It is believed that much of the rest of the human genome serves regulatory and structural functions. The exact number of genes carried by humans is not yet known, but is likely to be somewhere between 35,000 and 100,000.

Humans are more than 99% identical to each other at the DNA level. Only identical twins possess identical genomic DNA. It is the <1% variability in our DNA that makes each of us unique. In recent years, the variability in human DNA has been used as a fossil record to study human history and trace the migration of humans out of Africa as we spread across and populated the Earth. What we have learned from our DNA is clear: every human on the planet today is related to every other human. We all are descended from the same trunk of the human family tree: brothers and sisters separated only by time and geography.

The photograph in this slide is of twin stacks containing a total of eighty-nine Houston telephone books, spiraled to mimic the shape of the DNA double helix. Together the two stacks of telephone books contain approximately the same number of characters as the haploid human genome (23 chromosomes) contains base pairs.

DNA sequence variations are a normal part of the genetic composition of every living thing. Only identical twins share identical genomic DNA, but even identical twins can have differences in their DNA if sporadic mutations occur after twinning.

Changes in DNA are the basis for the adaptation, evolution, and speciation of living things. Variations in DNA also are the basis for susceptibility, or resistance, to genetically influenced disorders.