10 Генетическая инженерия (1160079), страница 4
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The clonecontaining the particular gene a researcher is interested in must beidentified within the thousands of clones in the library as describedbelow. If the desired DNA is from a mammal with a genome of 3 x 109base pairs of DNA and cosmids are used as cloning vectors, then thelibrary must contain about 350,000 recombinant cosmids, each with adifferent insert of 35,000 to 45,000 base pairs, for there to be a 99%chance that any desired gene of unique sequence is represented in thelibrary.
The fragments in a genomic library derived from a higher eukaryote include not only genes but the noncoding DNA that makes up alarge portion of many eukaryotic genomes.A more specialized and exclusive DNA library can be constructedso as to include only those genes that are expressed in a given organismor even in certain cells or tissues.
The critical difference between genesthat are expressed and genes that are not is that the former are transcribed into RNA. The mRNA from an organism or certain cells derivedfrom the organism is extracted, and complementary DNAs (cDNAs)are produced from the RNA in a multistep reaction catalyzed by reverse transcriptase (Fig. 28-9; Chapter 25). The resulting doublestranded DNA fragments are then inserted into a suitable vector andcloned, creating a population of clones called a cDNA library.
Amongthe first eukaryotic genes characterized were those encoding globins,primarily because cloning of these genes was facilitated by makingcDNA libraries from erythrocyte precursor cells, in which about half ofthe mRNA codes for globins.Hybridization Identifies Specific Sequencesin a DNA LibraryAfter a DNA library has been generated, the challenge is to isolate onegene of interest from among the millions of other DNA segments represented in a DNA library. Because each gene has a unique nucleotidesequence, it often can be detected with the aid of a labeled (e.g., radioactive) DNA fragment that is complementary to it, called a probe.Typically, nitrocellulose paper is pressed onto an agar plate containingmany individual bacterial colonies each containing a different recombinant DNA.
Some cells from each colony adhere to the paper, forming areplica of the plate. The paper is treated with alkali to disrupt the cellsand denature the DNA within, which remains localized to the regionaround the colony from which it came. The radioactive DNA probe isthen added to the paper, annealing only to DNA containing the complementary gene. The labeled colony is visualized by autoradiography(Fig. 28-10). Detecting any nucleic acid with a labeled nucleic acidprobe that is complementary to it is an application of nucleic acid hybridization (see Fig.
12-31), and many variations on this procedurehave been developed to detect both DNA (Box 28-1, p. 996) and RNA.995Chapter 28 Recombinant DNA TechnologyAgar plate withtransformedbacterial coloniesFigure 28-10 Identifying a clone with the desiredDNA segment; a schematic diagram of a hybridization method. The radioactive DNA probe hybridizesonly with homologous DNA. The annealed radioactive probe is revealed by autoradiography. Whenthe labeled colonies have been identified, the corresponding colonies on the original agar plate can beused as a source of cloned DNA for further study.Press nitrocellulose paper ontothe agar plate.
Some cells fromeach colony stick to the paper.Frequently, the limiting step in cloning a gene is finding or generating a complementary strand of nucleic acid to use as a probe. Theorigin of a probe depends on what is known about the gene under investigation. Sometimes a homologous gene cloned from another speciescan be used as a probe. Alternatively, if the protein product of a genehas been purified, probes can be designed and synthesized on the basisof its amino acid sequence and a knowledge of the genetic code (Fig.28-11).In this work, the synthesis and sequencing methods for proteinsand DNA described in previous chapters come together and complement one another.
Just as the sequence of an isolated protein can beused to isolate the corresponding gene, a cloned gene can be used toisolate its protein product. The sequence of an isolated gene can beused to design and chemically synthesize a short peptide that represents part of the protein product of the gene.
Antibodies to this synthetic peptide can then be used to identify and purify the protein itself(Chapter 6) for further study.Nitrocellulose paperTreat with alkali to disruptcells and expose denaturedDNA.DNA bound to paperRadiolabeled DNA probeIncubate the paper with theradiolabeled probe, then wash.Figure 28-11 Designing a probe to detect the genefor a protein of known amino acid sequence. Because the genetic code is degenerate, there is morethan one possible DNA sequence that codes for anygiven amino acid sequence.
As the correct DNAsequence cannot be known in advance, the probe isdesigned to be complementary to a region of thegene with minimal degeneracy. The oligonucleotidesare synthesized with the sequence selectively randomized so that some contain either of the two possible nucleotides at each position of potential degeneracy (shaded in red). In the example shown,the synthesized oligonucleotide is actually a mixture of eight different sequences; one of the eightwill complement the gene perfectly. All eight willmatch in at least 17 of 20 positions.Known amino acid sequence H3NPossible codons\ — Probe annealed tocolonies of interestExposex-ray filmto paper.Gly — Leu — Pro — Trp — Glu — Asp — Met — Trp — Phe — Val — ArgOUA CCA U G GGGCJ UUGcccGG|C C$$GG®(5')GG|CCGSyntheticprobesGAC AUG UGG UUC GUiA AGA(3')UUU GU|C AGGGATJGUftr GGAGU|G € G GCGURegion of minimal degeneracyCGGGAAGAGG AGA U QUU G G U l JCU20 nucleotides long, 8 possible sequencesCOO996Part IV Information PathwaysBOX 28-1A New Weapon in Forensic MedicineTraditionally, one of the most accurate methods forplacing an individual at the scene of a crime hasbeen a fingerprint.
A new identification techniquehas become available based on methods developedfor recombinant DNA technology. This is sometimes called DNA fingerprinting (also DNA typingor DNA profiling), and in some ways it is morepowerful than any other identification method.DNA fingerprinting is based on sequencepolymorphisms that occur in the human genome(and the genome of every other organism). Sequence polymorphisms are slight sequence differences (usually single base-pair changes) that occurfrom individual to individual once every few hundred base pairs, on average.
Each difference fromthe consensus human genome sequence is generally present in only a fraction of the human population, but every individual has some of them. Someof the sequence changes affect recognition sites forrestriction enzymes, resulting in variation fromindividual to individual in the size of certain DNAfragments produced by digestion with a particularrestriction enzyme.
These size differences are referred to as restriction fragment length polymorphisms, or RFLPs. A probe for a sequencethat is repeated several times in the human genome generally identifies a few of the thousands ofDNA fragments generated when the human genome is digested with a restriction endonuclease.The detection of RFLPs relies on a specializedhybridization procedure called Southern blotting (Fig. 1). DNA fragments from digestion ofgenomic DNA by restriction endonucleases arefirst separated according to size by electrophoresisin an agarose gel. The DNA fragments are denatured by soaking the gel in alkali, then transferredto nitrocellulose paper in such a way as to reproduce on the paper the distribution of fragments inthe gel.
The paper is then immersed in a solutioncontaining a radioactively labeled DNA probe.Fragments to which the probe hybridizes are revealed by autoradiography, using procedures similar to those described in Figure 28-10.The genomic DNA sequences used in these testsare generally regions containing repetitive DNA(short sequences repeated thousands of times intandem; see p. 797), which are common in the genomes of higher eukaryotes. The number of re-peated units in such DNA varies from individual toindividual (except in the case of identical twins).
Ifa suitable probe is chosen, the pattern of bands insuch an experiment can be distinctive for each individual tested. If several probes are used, the testcan be made so selective that it can positively identify a single individual in the human population.However, the Southern blot procedure requires relatively fresh DNA samples and larger amounts ofDNA than are generally present at a crime scene.To increase sensitivity, RFLP analysis is beingaugmented by polymerase chain reaction (PCR)methods (see Fig. 28-12), which permit vanishingly small amounts of DNA to be amplified. Theimproved tests allow DNA fingerprints to be obtained from a single hair, a small semen samplefrom a rape victim, or from samples that might bemonths or even many years old.Since their introduction in 1985, these methodshave been developed to the point where they areproving decisive in court cases worldwide.
In theexample in Figure 1, the DNA from a semen sample obtained from a rape and murder victim wasanalyzed along with DNA samples from the victimand two suspects. Each of the DNA samples wascleaved into fragments and separated by gel electrophoresis. Radioactive DNA probes were used toidentify a small subset of these fragments that contained sequences complementary to the probe.
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