Potential Use Of Dna In Nanoengineering

As a test case for nanoengineering with DNA, we have explored the use of DNA as a spacer of known length between two sites with biological functionality. The immuno-logical problem which motivated this work was a desire to understand the complex set of molecular interactions that occurs when an antigen-presenting cell is recognized by a T lymphocyte. Antigen is presented as peptide bound to a cell surface molecule known as a major histocompatibility molecule, in complex with associated...

Separation Of Dna In The Ultracentrifuge

Velocity or equilibrium ultracentrifugation of DNA represents the only serious alternative to DNA electrophoresis. For certain applications it is a powerful tool. However, for most applications, the resolution of ultracentrifugation just isn't high enough to compete with electrophoresis. DNA can be separated by size in the ultracentrifuge by zonal sedimentation. Commonly density gradients of small molecules like sucrose are employed to prevent convection caused by gravitational instabilities....

Cloning Dnas In Bacterial Plasmids And Bacteriophage

Cloning is the process of making many identical cells (or organisms) from a single precursor. If that precursor contains a target DNA molecule of interest, the cloning process will amplify that single molecule into a whole population. Most single-cell organisms are clonal. Their progeny are identical replicas. Cloning can also be done by manipulating single, immortal, or immortalizable cells of higher organisms including plants and animals. Here we concentrate on bacterial cloning, which can be...

ZE a2LE a2E f aJL aj

Thus from simple considerations we are led to the conclusion that the mobility of DNA in electrophoresis should be independent of size. Indeed this prediction was verified by the work of Norman Davidson and his collaborators more than 20 years ago. Electrophoresis of DNA in free solution fails to achieve any size fractionation at all. Why, then is elec-trophoresis such a powerful tool in fractionating DNA. The answers will all have to lie with the ways in which DNA molecules interact with gels...

Prospects For Electrophoretic Purification Of Chromosomes

In principle, it should be possible to use agarose gel electrophoresis to purify chromosomes. DNA molecules up to about 50,000 bp in size are well resolved by ordinary agarose electrophoresis while larger DNAs, up to about 10 Mb in size, can be fractionated effectively by pulsed field gel (PFG) electrophoresis. Secondary pulsed electrophoresis (SPFG), where short intense pulses are superimposed on the normally slowly varying pulses in PFG, expands the fractionation range of DNA even further...

Chromosome Purification

The past decade has seen tremendous strides in our ability to purify specific human chromosomes. Early attempts, using density gradient sedimentation, never achieved the sort of resolution necessary to become a routine analytical or preparative technique. The key advance was the creation of fluorescence activated flow sorters with sufficient intensity to allow accurate fluorescence determinations on single metaphase chromosomes. The fluorescence activated flow sorter originally was developed...

Methylated Bases

DNA from most higher organisms and from many lower organisms have additional analogues of the normal bases. In bacteria these are principally M> -methyl A and 5-methyl C (or 4-methyl C). Higher organisms contain 5-methyl C. The presence of methylated bases has a strong biological effect. Once in place, the methylated bases are apt to maintain their methylated status after DNA replication. This is because the hemi-methylated duplex produced by one round of DNA synthesis is a far better...

More On Mitochondrial

The pure maternal inheritance of mitochondria makes it very easy to trace lineages in human populations, since all of the complexities of diploid genetics are avoided. The only analogous situation is the Y chromosome which must be paternally inherited. One region of the mitochondrial DNA, near the replication origin, codes for no known genes. This region shows a relatively fast rate of evolution. By monitoring the changes in the DNA of this region, Allen Wilson and his coworkers have attempted...

Simple Enzymatic Manipulation Of Dnas

The structure of a DNA strand is an alternating polymer of phosphate and sugar-based units called nucleosides. Thus the ends of the chain can occur at phosphates (p) or at sugar hydroxyls (OH). Polynucleotide kinase can specifically add a phosphate to the 5'-end of a DNA chain. 5' HO-ApTpCpG-OH 3' 5' pApTpCpG-OH 3' Phosphatases remove phosphates from one or both ends. 5' pApTpCpGp 3' alkaline ' 5' HO-ApTpCpG-OH 3' DNA ligases will join together two DNA strands that lie adjacent along a...

Basic Strategy For Genetic Analysis In The Human Linkage Mapping

In most organisms, genetics is carried out by breeding specific pairs of parents and examining the characteristics of their offspring. Clearly this approach is not practical in the human. Even leaving aside the tremendous ethical issues such an approach would raise, the small size of our families, and the long lifespan of our species, make genetic experimentation all but impossible. Instead, what must be done is to perform retrospective analyses of inheritance in families. Statistical analysis...

Current State Of The Human Genetic

Several major efforts to make genetic maps of the human genome have occurred during the past few years, and recent emphasis has been on merging these efforts to forge consensus maps. A few examples of the status of these maps several years ago are given in Figure 6.29a and b. These are sex-averaged maps because they have a larger density of markers. The ideal framework map will have markers spaced uniformly at distances around 3 to 5 cM, since this is the most efficient sort of map to use to...

Twostage Model Of Recombination

For recombination to occur, homologous chromosomes must pair up with their DNA sequences aligned. Studies in yeast have led to a rather complex model for this process. The complications occur because DNA sequence similarities or identities occur not only between the pairs of homologs but also between different chromosomes as a result of dispersed repeated DNA sequences (see Chapter 14) or dispersed gene families with similar or identical members. The recombination apparatus in bacteria, yeast,...

Bacterial colonies

And release bacteriophage, then a convenient method of cloning is to grow a continuous layer of susceptible bacteria and then cover the culture with a thin layer of dilute bacteriophage suspension. Individual bacteriophage will infect cells, leading to continual cycles of bacteriophage growth and cell lysis until visible holes in the bacterial lawn, called bacteriophage plaques, are observed. Individual colonies or plaques are selected for further study by picking, literally by sampling with a...

Detection Of Homozygous Regions

Regions Homozygosity

Because the human species is highly outbred, homozygous regions are rare. Such regions can be found, however, by traditional methods or by some of the fairly novel methods that will be described in Chapter 13. Homozygous regions are very useful both in the diagnosis of cancer and in certain types of genetic mapping. The significance of homozygous regions in cancer is shown in Figure 6.37. Oncogenes are ordinary cellular genes, or foreign genes that under appropriate circumstances can lead to...

Good amplification after first few cycles

Single Sided Pcr

Figure 4.6 Introducing extra functionalities by appropriately designed primers. (a) Incorporation of a 5'-terminal label (asterisk). (b) Incorporation of flanking restriction sites, useful for subsequent cloning. (c) Compensating for less than optimal length initial sequence information. This problem typically arises when one is trying to walk by from a known region of the genome into flanking unknown regions. Then one starts with a bit of known sequence at the extreme edge of the charted...

Thermodynamics Of Imperfectly Paired Duplexes

In contrast to the small number of discrete interactions that must be considered in calculating the energetics of perfectly paired DNA duplexes, there is a plethora of ways that duplexes can pair imperfectly. We have available model compound data on most of these so that estimates of the energetics can be made. However, the large number of possibilities precludes a complete analysis of imperfections in the context of all possible sequences, at least for the present. The simplest imperfection is...

Subchromosomal Mapping Panels

Frequently it is possible to assemble sets of cell lines containing fragments of only one chromosome of interest. The occurrences of a DNA probe sequence in a subset of these cell lines can be used to assign the probe to a specific region of the chromosome. The accuracy of that assignment is determined by the accuracy with which the particular chromosome fragments contained in each cell line are known. This can vary quite considerably, but in general this approach is quite a powerful and...

Basic Principles Of The Polymerase Chain Reaction

What makes PCR a tool of immense power and flexibility is the requirement of DNA polymerases for pre-existing DNA primers. Thus DNA polymerases cannot start DNA chains de novo a primer can be used to determine where, along a DNA template, the synthesis of the complement of that stand begins. It is this primer requirement that allows the selective amplification of any DNA region by using appropriate, specific DNA primers. Once started, a DNA polymerase like E. coli DNA polymerase I pol I will...

Double Helical Structure

The two common base pairs A-T and G-C are well-known, and little evidence for other base interactions within the DNA's double helix exists. The key feature of double-helical DNAs duplexes , which dominates their properties, is that an axis of symmetry relates the two strands Box 1.1 . Figure 1.1 Structure of the phosphodiester backbone of DNA and RNA, and the four major bases found in DNA and RNA the purines, adenine A and guanine G , and the pyrimidines, cytosine C and thymine T or uracil U ....

C2 Symmetry

Hairpin Helix Single Stranded Dna

C2 symmetry implies that a structure is composed of two identical parts. An axis of rotation can be found that interchanges these two parts by a 180-degree rotation. This axis is called a C2 axis. An example of a common object that has such symmetry is the stitching of a baseball, which is used to bring two figure-8 shaped structures together to make a spherical shell. An example of a well-known biological structure with C2 symmetry is the hemoglobin tetramer which is made up of two identical...

Pcr Noise Mispriming

Typical PCR conditions with two convergent primers offer a number of possible unintended primed amplifications. These are illustrated in Figure 4.4. If the primers are not chosen wisely, one of the two primers may be able to act alone to amplify DNA as shown in Figure 4.4b. Alternatively, the two convergent primers may have more than one site in the target that allows amplification. There is no way to plan for these events, unless the entire sequence of the sample is known. However, the chances...

Unusual Characteristics Of Sex Chromosomes And Mitochondria

In mammals a female carries two copies of the X chromosome males have one X and one Y. However, this simple difference in karyotype the set of chromosomes has profound effects that go beyond just the establishment of sex. The first thing to consider is why we need sex at all. In species with just one sex, each organism can reproduce clon-ally. The offspring of that organism may be identical. If the organism inhabits a wide ecological range, different selection processes will produce a...

Physical Mapping

WHY HIGH-RESOLUTION PHYSICAL MAPS ARE NEEDED Physical maps are needed because ordinary human genetic maps are not detailed enough to allow the DNA that corresponds to particular genes to be isolated efficiently. Physical maps are also needed as the source for the DNA samples that can serve as the actual substrate for large-scale DNA sequencing projects. Genetic linkage mapping provides a set of ordered markers. In experimental organisms this set can be almost as dense as one wishes. In humans...

Quantitative Highresolution Fish

Fiber Fish With Gene Specific Probe

The quantitative analysis of distances in high-resolution FISH has been pioneered by two groups headed by Barb Trask, currently at the University of Washington, and Jeanne Lawrence, at the University of Massachusetts in Amherst. Others have learned their methods and begun to practice them. A few representative analyses are shown in Figures 7.18 and 7.19. The distribution of measured distances between two fixed markers in individual samples of interphase chromatin varies over quite a wide range,...

Padlock Probes

Ulf Landegren and his collaborators recently demonstrated that DNA topology can be used to improve the stringency of detection of correctly hybridized DNA probes. A schematic view of the procedure that they have developed is shown in Figure 3.20. Consider the hybridization of a linear single-stranded probe to a single-stranded circular target. The probe is specially designed so that it's 3'- and 5'-terminal sequences are complementary to a continuous block of DNA sequence in the target. Probes...