Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 66
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A gel is a complex molecular network that contains narrow, tortuous passages,so smaller DNA molecules pass through more easily; hence the rate of movement increases as the molecularweight decreases. For each molecule, the rate of movement depends primarily on the molecular size, provided themolecule is linear and not too large. Figure 5.34 shows the result of electrophoresis of a collection of doublestranded DNA molecules in an agarose gel.
Each discrete region containing DNA is called a band.The Southern BlotSeveral techniques enable a researcher to locate a particular DNA fragment in a gel. One of the most generallyapplicable procedures is the Southern blot. In this procedure, a gel in which DNA molecules have been separatedby electrophoresis is treated with alkali to denature the DNA and render it single-stranded. Then the DNA istransferred to a sheet of nitrocellulose in such a way that the relative positions of the DNA bands are maintained(Figure 5.35).
The nitrocellulose, to which the single-stranded DNA binds tightly, is then exposed to denaturedradioactive complementary RNA orFigure 5.34Gel electrophoresis of DNA. Molecules ofdifferent sizes were mixed and placed in a well.Electrophoresis was in the vertical direction.The DNA has been made visible by the additionof a dye (ethidium bromide) that binds only toDNA and that fluoresces when the gel isilluminated with short-wavelength ultraviolet light.DNA (the probe) in a way that leads the complementary strands to anneal to form duplex molecules.
Radioactivitybecomes stably bound (resistant to removal by washing) to the DNA only at positions atPage 207Figure 5.35Southern blot. (A) DNA restriction fragments are separated by electrophoresis, blotted from the gel onto a nitrocellulose or nylonfilter, and chemically attached by the use of ultraviolet light. (B) The strands are denatured and mixed with radioactiveprobe DNA, which binds with complementary sequences present on the filter. The bound probe remains, whereas unboundprobe washes off. (C) Bound probe is revealed by darkening of photographicfilm placed over the filter. The positions of the bandsindicate which restriction fragments contain DNA sequences homologous with those in the probe.which base sequences complementary to the radioactive molecules are present, so that duplex molecules can form.The radioactivity is located by placing the paper in contact with x-ray film; after development of the film,blackened regions indicate positions of radioactivity.
For example, a cloned DNA fragment from one species maybe used as probe DNA in a Southern blot with DNA from another species; the probe will hybridize only withrestriction fragments containing DNA sequences that are complementary enough to allow stable duplexes to form.5.8—The Polymerase Chain ReactionIt is also possible to obtain large quantities of a particular DNA sequence merely by selective replication. Themethod for selective replication is called the polymerase chain reaction (PCR), and it uses DNA polymerase anda pair of short, synthetic oligonucleotide primers, usually about 20 nucleotides in length, that are complementaryin sequence to the ends of any DNA sequence of interest.
Starting with a mixture containing as little as onemolecule of the fragment of interest, repeated rounds of DNA replication increase the number of moleculesexponentially. For example, starting with a single molecule, 25 rounds of DNA replication will result in 225 = 3.4 ×107 molecules. This number of molecules of the amplified fragment is so much greater than that of the otherunamplified molecules in the original mixture that the amplified DNA can often be used without furtherpurification.
For example, a single fragment of 3000 base pairs in E. coli accounts for only 0.06 percent of the totalDNA in thisPage 208organism. However, if this single fragment were replicated through 25 rounds of replication, then 99.995 percent ofthe resulting mixture would consist of the amplified sequence.An outline of the polymerase chain reaction is shown in Figure 5.36. The DNA sequence to be amplified and theoligonucleotide sequences are shown in contrasting colors. The oligonucleotides act as primers for DNAreplication, because they anneal to the ends of the sequence to be amplified and become the substrates for chainelongation by DNA polymerase. In the first cycle of PCR amplification, the DNA is denatured to separate thestrands.
The denaturation temperature is usually around 95°C. Then the temperature is decreased to allowannealing in the presence of a vast excess of the primer oligonucleotides. The annealing temperature is typically inthe range from 50°C to 60°C, depending largely on the G + C content of the oligonucleotide primers.
Thetemperature is raised slightly, to about 70°C, for the elongation of each primer. The first cycle in PCR producestwo copies of each molecule containing sequences complementary to the primers. The second cycle of PCR issimilar to the first. The DNA is denatured, then renatured in the presence of an excess of primer oligonucleotides,and the primers are elongated by DNA polymerase; after this cycle, there are four copies of each molecule presentin the original mixture. The steps of denaturation, renaturation, and replication are repeated from 20–30 times, andin each cycle, the number of molecules of the amplified sequence is doubled.
The theoretical result of 25 rounds ofamplification is 225 copies of each template molecule present in the original mixture.Implementation of PCR with conventional DNA polymerase is not practical, because at the high temperaturenecessary for denaturation, the polymerase is itself irreversibly unfolded and becomes inactive. However, DNApolymerase isolated from certain archaebacteria is heat stable because the organisms normally live in hot springs attemperatures well above 90°C, such as are found in Yellowstone National Park. Such organisms are said to bethermophiles.
The most widely used heat-stable DNA polymerase is called Taq polymerase, because it wasoriginally isolated from the thermophilic archaebacterium Thermus aquaticus.Figure 5.37 presents a closer look at the primer oligonucleotide sequences and their relationship to the DNAsequence to be amplified. The primers are designed to anneal to opposite DNA strands at the extreme ends of theregion to be amplified. The primers are oriented with their 3' ends facing the sequence to be amplified so that, inDNA synthesis, the new strands grow toward each other but along complementary template strands.
In this way,each newly synthesized strand terminates in a sequence that can anneal with the complementary primer and so canbe used for further amplification.PCR amplification is very useful for generating large quantities of a specific DNA sequence. The principallimitation of the technique is that the DNA sequences at the ends of the region to be amplified must be known sothat primer oligonucleotides can be synthesized.
In addition, sequences longer than about 5000 base pairs cannot bereplicated efficiently by conventional PCR procedures. On the other hand, there are many applications in whichPCR amplification is useful. PCR can be employed to study many different mutant alleles of a gene whosewildtype sequence is known in order to identify the molecular basis of the mutations.
Similarly, DNA sequencevariation among alleles present in natural populations can easily be determined by using PCR. The PCR procedurehas also come into widespread use in clinical laboratories for diagnosis. To take just one very important example,the presence of the human immunodeficiency virus (HIV), which causes acquired immune deficiency syndrome(AIDS), can be detected in trace quantities in blood banks via PCR by using primers complementary to sequencesin the viral genetic material. These and other applications of PCR are facilitated by the fact that the procedure lendsitself to automation—for example, by the use of mechanical robots to set up the reactions.Page 209Figure 5.36Polymerase chain reaction (PCR) for amplification of particular DNA sequences.
Oligonucleotide primers (green) that arecomplementary to the ends of the sequence of interest (blue) are used in repeated rounds of denaturation, annealing, and DNAreplication.Copies of the target sequence are shown in pink. The number of copies of the target sequence doubles in each roundof replication,eventually overwhelming any other sequences that may be present.Page 210Figure 5.37Example of primer sequences used in PCR amplification Newly synthesizedDNA is shown in pink (A) Original DNA duplex (blue) and primers (green).(B) The original DNA duplex is denatured and the primers annealed.
(C) Afterone round of binding of the primers and elongation. Each primer has beenextended at the 3' end. (D) After another round of replication Each primer is againextended at the 3' end, but the elongation terminates at the 5' end of the primeroligonucleotide on the other strand After the first round of replication, eachnewly synthesized DNA molecule forms a new template. so the number ofcopies increases exponentially.5.9—Determination of the Sequence of Bases in DNAA great deal of information about gene structure and gene expression can be obtained by direct determination ofthe sequence of bases in a DNA molecule. Several techniques are available for base sequencing; the most widelyused method is described in this section. No technique can determine the sequence of bases in an entirechromosome in a single experiment, so chromosomes are first cut into fragments a few hundred base pairs long, asize that can be sequenced easily.
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