10 Генетическая инженерия (1160079), страница 5
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Thesizes of the fragments identified varied from oneindividual to the next, as seen here in the differentpatterns for the three individuals (victim and twosuspects) tested. One rape suspect's DNA exhibitsa banding pattern identical to that of a semen sample taken from the victim. One probe was usedhere, but three or four different probes would beused to make a positive identification. Resultshave been used to help both convict and acquit suspects. DNA fingerprints can also be used to establish paternity with an extraordinary degree of certainty.
The results of DNA fingerprinting havebeen successfully challenged in some court casesbecause of irregularities and a lack of controls inmany early examples. The far-reaching impact ofthis technology on court cases will neverthelesscontinue to grow as standards are agreed upon andthe methods become widely established in forensiclaboratories.Chapter 28 Recombinant DNA TechnologyChromosomal DNA(e.g., Suspect 1)Cleave with restrictionendonucleases.DNA fragmentsSeparate fragments by agarosegel electrophoresis (unlabeled).#Figure 1 The Southern blot procedure, as appliedto DNA fingerprinting.11Illliikiiilliikiiiliis1 I-illllllliiliillllmmIncubatewithprobe,thenwash.mm —sS111--Si1=ii iUlllliiliiis s~ 11sii | "WHIN8mm§1s i™" sS111i-11i-iiiiiiiig 255IDenatureDNA, andtransfer tonitrocellulosepaper.ifin nil«i—Isiiiiii55I ••&- 5•» Xiiiiiii--Iim|iiiiiiiI I_|Mii•j1" • 1 I I"li i •• z1s i I*— s5s ss• - i1 IiiiiiiiIlllIIM15 I |i -zii s SIlll8mili iuuruur urU ururiiiiiiniRadiolabeledDNA probeExposex-rayfilmto paper.997Part IV Information Pathways998Region of target DNAto be amplified1I(T) Heat to separatestrands.(2) Cool; add syntheticoligonucleotide primersi(3) Add thermostable DNApolymerase to catalyze5' —> 3' DNA synthesis.iliFigure 28-12 Amplifying a specific DNA segmentwith a polymerase chain reaction.
DNA strands areseparated by heating, then annealed to an excess ofshort synthetic DNA primers (blue) that flank theregion to be amplified. After polymerization, theprocess is repeated for 25 or 30 cycles. The thermostable DNA polymerase Taq\ (from Thermus thermophilus, bacteria that grow in hot springs) is notdenatured by the heating steps.Specific DNA Sequences Can Be AmplifiedRepeat steps (T) and (2).DNA synthesis (step (3) )is catalyzed by thethermostable DNApolymerase (still present).Repeat steps (T)through (3).If one knows the sequence of at least part of a DNA segment to becloned, cloning can be facilitated by amplifying the DNA segment in aprocess called a polymerase chain reaction (PCR), invented byKary Mullis in 1984.
Two oligonucleotides are synthesized, each complementary to a short sequence in one strand of the desired DNA segment and positioned just beyond the end of the sequence to be amplified; these synthetic oligonucleotides can be used as primers forreplication of the DNA segment in vitro (Fig. 28-12). Isolated DNAcontaining the segment to be cloned is heated briefly to denature it,then cooled in the presence of a large excess of the synthetic oligonucleotide primers.
A heat-stable DNA polymerase called Taql and the fourdeoxynucleoside triphosphates are then added, and the primed DNAsegment is selectively replicated. This process is repeated through 25or 30 cycles, which can take only a few hours when automated, amplifying the DNA segment to the point where it can be readily isolatedand cloned.The PCR method is sensitive enough to detect as little as one DNAmolecule in almost any type of sample.
It has been used to clone DNAfragments from mummies and the remains of extinct animals such asthe woolly mammoth, creating the new fields of molecular archaeologyand molecular paleontology. In addition to its usefulness for cloningDNA, it is a potent new tool in forensic medicine (Box 28-1). It is alsobeing used for detection of viral infections before they cause symptomsor elicit a detectable immune response and in prenatal diagnosis of awide array of genetic diseases.The Products of Recombinant DNA TechnologyAfter 25 cycles, the target sequence hasbeen amplified about 106 -fold.Normally, cloning a gene is only the first step in a much grander design. A cloned gene can be used to generate large amounts of its proteinproduct.
The amino acid sequence of the protein can be altered by introducing base-pair changes in the gene, a strategy that can be verypowerful in addressing questions concerning protein folding, structure,and function. Increasingly sophisticated methods for moving DNA intoand out of cells of all types are providing another avenue for studyinggene function and regulation, and are allowing the introduction of newtraits into plants and animals.Our focus now turns to applications of DNA cloning, beginningwith the proteins produced by cloned genes. We then describe cloningprocedures used for a variety of eukaryotic cells, before finishing withan overview of the potential and implications of this technology.1002Part IV Information Pathwaysmation of this type in which the introduced DNA is integrated into acellular chromosome is called integrative transformation; it occursat low frequency.Transformation efficiencies can be increased by introducing clonedDNA on a self-replicating plasmid.
A naturally occurring yeast plasmid called the 2 micron (2/JL) plasmid has been engineered to create avariety of cloning vectors that incorporate a replication origin andother sequences needed for plasmid maintenance in yeast. Anothertype of plasmid that provides an increase in transformation frequencies contains a yeast chromosomal origin of replication (an autonomously replicating sequence, ARS; see p. 830). Such plasmids aresomewhat unstable (and are lost from the yeast population) unlessthey also contain a centromere that permits them to function and segregate like yeast chromosomes during cell division.Recombinant plasmids are available that incorporate multiplegenetic elements (replication origins, etc.), allowing them to be maintained in more than one species; for example, yeast or E.
coli. Plasmidsthat can be propagated in cells of two or more different species arecalled shuttle vectors.Cloning in Plants Is Aided by a Bacterial ParasiteThe introduction of recombinant DNA into plants has enormous potential for agriculture, producing more nutritious and higher-yieldingcrops that are resistant to environmental stresses such as insect pests,disease, cold, and drought. Unlike animals, fertile plants of some species may be generated from a single transformed cell.
Thus, a geneintroduced into a plant cell may ultimately be transmitted to progenythrough seed in successive generations. As with all systems, cloning inplant cells has its own peculiar problems. No naturally occurring plasmids have been found in plants to facilitate this process, and thus themost challenging task is getting DNA into plant cells.Fortunately, scientists have found an important and adaptableally in the soil bacterium Agrobacterium tumefaciens.
The bacteriuminvades plants at the site of a wound, transforming plant cells near thewound and inducing them to form a tumor called a crown gall. Agrobacterium contains a large (—200,000 base pair) plasmid called the Tiplasmid (Fig. 28-15). When the bacterium contacts a plant cell, asegment of this plasmid, called the T DNA (—23,000 base pairs), istransferred from the Ti plasmid to the plant cell nucleus and is integrated at a random position in one of the plant's chromosomes duringtransformation. This is a rare example of DNA transfer from a prokaryote to a eukaryote; it represents a natural genetic engineeringprocess.The T DNA encodes enzymes that convert plant metabolites to twoclasses of compounds important to the bacterium (Fig.
28-16). Thefirst class consists of the plant growth hormones, auxins andcytokinins, which stimulate growth of the transformed plant cells toform the crown gall tumor. The second is a series of unusual aminoacids called opines, a food source for the bacteria. The opines are produced at high concentrations in the tumor and secreted to the surroundings. They can only be metabolized by Agrobacterium, using enzymes encoded elsewhere on the Ti plasmid. In this manner thebacteria monopolize available nutrients by converting them to a formthat does not benefit any other organism.Chapter 28 Recombinant DNA Technologyvir genesFigure 28-15 (a) The Ti (tumor-inducing) plasmid of Agrobacterium tumefaciens.
(b) Acetosyringone, a phenolic compound produced by plants, increases in concentration in wounded plant cells andis released from the cells. Agrobacterium sensesthis compound, and the virulence (vir) genes on theTi plasmid are expressed. The vir genes encodeenzymes needed to introduce the T DNA into thegenome of plant cells in the vicinity of the wound.A single-stranded copy of the T DNA is synthesizedand transferred to the plant cell, where it is converted to duplex DNA and integrated into a chromosome. The T DNA encodes enzymes that synthesize growth hormones and opines (see Fig. 28-16)from common metabolites.
Opines can be metabolized only by Agrobacterium, which uses them as anutrient source. Expression of the T DNA genes bytransformed plant cells thus leads to cell growth(tumor formation) and the diversion of plant cellnutrients to the invading bacteria.TDNATi plasmid(a)Agrobacterium cellWounded plantcell producesacetosyringoneAcetosyringoneactivates vir genesSynthesis of auxins,cytokinins, opines; ^_tumor formationCopy of T DNA istransferred and integratedinto a random site in a plantchromosomeFigure 28-16 Metabolites produced in Agrobacterium -infected plant cells.
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