what technique would a scientist use to produce many copies of a desired piece of dna

Until the early 1970s Deoxyribonucleic acid was the most difficult cellular molecule for the biochemist to clarify. Enormously long and chemically monotonous, the string of nucleotides that forms the genetic material of an organism could be examined only indirectly, by poly peptide or RNA sequencing or past genetic analysis. Today the situation has changed entirely. From beingness the nearly difficult macromolecule of the jail cell to clarify, DNA has become the easiest. Information technology is now possible to isolate a specific region of a genome, to produce a virtually unlimited number of copies of it, and to determine the sequence of its nucleotides overnight. At the top of the Man Genome Projection, large facilities with automated machines were generating DNA sequences at the rate of 1000 nucleotides per second, effectually the clock. By related techniques, an isolated gene can be altered (engineered) at will and transferred back into the germ line of an animal or found, so every bit to become a functional and heritable part of the organism's genome.

These technical breakthroughs in genetic engineering—the ability to manipulate DNA with precision in a test tube or an organism—have had a dramatic impact on all aspects of prison cell biology by facilitating the study of cells and their macromolecules in previously unimagined ways. They accept led to the discovery of whole new classes of genes and proteins, while revealing that many proteins have been much more highly conserved in development than had been suspected. They have provided new tools for determining the functions of proteins and of individual domains inside proteins, revealing a host of unexpected relationships betwixt them. By making available big amounts of any protein, they take shown the way to efficient mass production of protein hormones and vaccines. Finally, by assuasive the regulatory regions of genes to be dissected, they provide biologists with an of import tool for unraveling the complex regulatory networks by which eucaryotic gene expression is controlled.

Recombinant Dna technology comprises a mixture of techniques, some new and some borrowed from other fields such every bit microbial genetics (Table viii-seven). Cardinal to the technology are the following key techniques:

Table 8-vii

Some Major Steps in the Development of Recombinant DNA and Transgenic Technology.

1.

Cleavage of DNA at specific sites past restriction nucleases, which greatly facilitates the isolation and manipulation of individual genes.

2.

DNA cloning either through the use of cloning vectors or the polymerase chain reaction, whereby a single DNA molecule tin can exist copied to generate many billions of identical molecules.

three.

Nucleic acid hybridization, which makes information technology possible to observe a specific sequence of Dna or RNA with great accuracy and sensitivity on the basis of its ability to bind a complementary nucleic acid sequence.

4.

Rapid sequencing of all the nucleotides in a purified Deoxyribonucleic acid fragment, which makes it possible to identify genes and to deduce the amino acid sequence of the proteins they encode.

five.

Simultaneous monitoring of the expression level of each gene in a cell, using nucleic acid microarrays that allow tens of thousands of hybridization reactions to be performed simultaneously.

In this affiliate nosotros describe each of these basic techniques, which together have revolutionized the written report of cell biology.

Large Deoxyribonucleic acid Molecules Are Cut into Fragments past Restriction Nucleases

Unlike a protein, a cistron does not be equally a discrete entity in cells, but rather as a small region of a much longer DNA molecule. Although the DNA molecules in a cell can be randomly broken into pocket-sized pieces by mechanical force, a fragment containing a single factor in a mammalian genome would even so be only one amidst a hundred thousand or more DNA fragments, duplicate in their average size. How could such a gene be purified? Because all Deoxyribonucleic acid molecules consist of an approximately equal mixture of the same four nucleotides, they cannot be readily separated, as proteins can, on the ground of their different charges and binding backdrop. Moreover, fifty-fifty if a purification scheme could be devised, vast amounts of Deoxyribonucleic acid would be needed to yield enough of any particular gene to exist useful for further experiments.

The solution to all of these problems began to emerge with the discovery of restriction nucleases. These enzymes, which tin be purified from bacteria, cut the Deoxyribonucleic acid double helix at specific sites defined by the local nucleotide sequence, thereby cleaving a long double-stranded DNA molecule into fragments of strictly defined sizes. Different brake nucleases have different sequence specificities, and it is relatively simple to find an enzyme that can create a DNA fragment that includes a particular gene. The size of the DNA fragment tin then be used as a basis for partial purification of the gene from a mixture.

Dissimilar species of bacteria make different restriction nucleases, which protect them from viruses past degrading incoming viral DNA. Each nuclease recognizes a specific sequence of 4 to eight nucleotides in DNA. These sequences, where they occur in the genome of the bacterium itself, are protected from cleavage by methylation at an A or a C rest; the sequences in foreign Deoxyribonucleic acid are generally non methylated and so are cleaved past the restriction nucleases. Large numbers of restriction nucleases have been purified from various species of bacteria; several hundred, near of which recognize different nucleotide sequences, are now available commercially.

Some restriction nucleases produce staggered cuts, which get out short single-stranded tails at the 2 ends of each fragment (Figure 8-21). Ends of this type are known as cohesive ends, as each tail can grade complementary base pairs with the tail at any other end produced by the aforementioned enzyme (Figure 8-22). The cohesive ends generated by brake enzymes permit any two DNA fragments to be easily joined together, as long every bit the fragments were generated with the same restriction nuclease (or with another nuclease that produces the same cohesive ends). Deoxyribonucleic acid molecules produced by splicing together 2 or more Dna fragments are called recombinant Deoxyribonucleic acid molecules; they have made possible many new types of prison cell-biological studies.

Figure 8-21

The Dna nucleotide sequences recognized by four widely used restriction nucleases. As in the examples shown, such sequences are oft six base pairs long and "palindromic" (that is, the nucleotide sequence is the aforementioned if the helix is turned (more than...)

Figure eight-22

Restriction nucleases produce Deoxyribonucleic acid fragments that can be hands joined together. Fragments with the same cohesive ends can readily join by complementary base of operations-pairing between their cohesive ends, as illustrated. The ii Dna fragments that bring together in this example (more...)

Gel Electrophoresis Separates DNA Molecules of Unlike Sizes

The length and purity of Dna molecules can be accurately determined past the same types of gel electrophoresis methods that accept proved so useful in the analysis of proteins. The procedure is really simpler than for proteins: considering each nucleotide in a nucleic acrid molecule already carries a single negative charge, in that location is no need to add the negatively charged detergent SDS that is required to make poly peptide molecules motion uniformly toward the positive electrode. For DNA fragments less than 500 nucleotides long, specially designed polyacrylamide gels let separation of molecules that differ in length by as little as a single nucleotide (Figure 8-23A). The pores in polyacrylamide gels, yet, are as well small to allow very large Deoxyribonucleic acid molecules to pass; to split up these by size, the much more porous gels formed by dilute solutions of agarose (a polysaccharide isolated from seaweed) are used (Effigy 8-23B). These DNA separation methods are widely used for both belittling and preparative purposes.

Figure eight-23

Gel electrophoresis techniques for separating Deoxyribonucleic acid molecules by size. In the three examples shown, electrophoresis is from top to lesser, so that the largest—and thus slowest-moving—DNA molecules are near the acme of the gel. In (A) a polyacrylamide (more...)

A variation of agarose gel electrophoresis, called pulsed-field gel electrophoresis, makes it possible to separate even extremely long DNA molecules. Ordinary gel electrophoresis fails to separate such molecules considering the steady electrical field stretches them out so that they travel end-first through the gel in snakelike configurations at a rate that is independent of their length. In pulsed-field gel electrophoresis, past contrast, the direction of the electrical field is changed periodically, which forces the molecules to reorient earlier continuing to motion snakelike through the gel. This reorientation takes much more time for larger molecules, so that longer molecules move more slowly than shorter ones. As a upshot, even entire bacterial or yeast chromosomes divide into discrete bands in pulsed-field gels and so can be sorted and identified on the ground of their size (Effigy 8-23C). Although a typical mammalian chromosome of 10^eight base of operations pairs is too large to exist sorted even in this fashion, large segments of these chromosomes are readily separated and identified if the chromosomal Dna is first cut with a restriction nuclease selected to recognize sequences that occur only rarely (in one case every ten,000 or more nucleotide pairs).

The DNA bands on agarose or polyacrylamide gels are invisible unless the Deoxyribonucleic acid is labeled or stained in some style. One sensitive method of staining Deoxyribonucleic acid is to betrayal it to the dye ethidium bromide, which fluoresces under ultraviolet light when information technology is spring to Dna (meet Figures 8-23B,C). An even more sensitive detection method incorporates a radioisotope into the Deoxyribonucleic acid molecules before electrophoresis; ³²P is often used equally it can be incorporated into DNA phosphates and emits an energetic β particle that is easily detected by autoradiography (as in Effigy eight-23A).

Purified DNA Molecules Can Be Specifically Labeled with Radioisotopes or Chemic Markers in vitro

Two procedures are widely used to characterization isolated DNA molecules. In the first method a DNA polymerase copies the Dna in the presence of nucleotides that are either radioactive (usually labeled with ³²P) or chemically tagged (Figure eight-24A). In this fashion "DNA probes" containing many labeled nucleotides can be produced for nucleic acid hybridization reactions (discussed below). The second procedure uses the bacteriophage enzyme polynucleotide kinase to transfer a single ³²P-labeled phosphate from ATP to the 5′ end of each DNA chain (Figure eight-24B). Because only one ³²P cantlet is incorporated by the kinase into each Dna strand, the Deoxyribonucleic acid molecules labeled in this mode are often non radioactive enough to be used as DNA probes; because they are labeled at but 1 end, however, they have been invaluable for other applications including Dna footprinting, as nosotros see shortly.

Figure 8-24

Methods for labeling Dna molecules in vitro. (A) A purified Dna polymerase enzyme labels all the nucleotides in a Dna molecule and can thereby produce highly radioactive Deoxyribonucleic acid probes. (B) Polynucleotide kinase labels but the 5′ ends of Dna strands; (more than...)

Today, radioactive labeling methods are being replaced past labeling with molecules that tin can exist detected chemically or through fluorescence. To produce such nonradioactive DNA molecules, particularly modified nucleotide precursors are used (Figure 8-24C). A Deoxyribonucleic acid molecule made in this mode is allowed to bind to its complementary Deoxyribonucleic acid sequence by hybridization, as discussed in the next section, and is and then detected with an antibody (or other ligand) that specifically recognizes its modified side chain (encounter Figure 8-28).

Effigy 8-28

Here, 6 different Deoxyribonucleic acid probes accept been used to mark the location of their respective nucleotide sequences on human being chromosome v at metaphase. The probes have been chemically labeled and detected with fluorescent antibodies. Both copies of chromosome (more...)

Nucleic Acid Hybridization Reactions Provide a Sensitive Way of Detecting Specific Nucleotide Sequences

When an aqueous solution of DNA is heated at 100°C or exposed to a very high pH (pH ≥ xiii), the complementary base pairs that normally hold the 2 strands of the double helix together are disrupted and the double helix speedily dissociates into two single strands. This process, called DNA denaturation, was for many years thought to exist irreversible. In 1961, nonetheless, it was discovered that complementary unmarried strands of DNA readily re-form double helices by a procedure called hybridization (also chosen DNA renaturation) if they are kept for a prolonged menstruation at 65°C. Similar hybridization reactions can occur between any ii single-stranded nucleic acid chains (Dna/Dna, RNA/RNA, or RNA/Deoxyribonucleic acid), provided that they have complementary nucleotide sequences. These specific hybridization reactions are widely used to discover and characterize specific nucleotide sequences in both RNA and Dna molecules.

Single-stranded Dna molecules used to notice complementary sequences are known as probes; these molecules, which carry radioactive or chemical markers to facilitate their detection, can exist anywhere from fifteen to thousands of nucleotides long. Hybridization reactions using Deoxyribonucleic acid probes are and so sensitive and selective that they can detect complementary sequences present at a concentration as low as one molecule per jail cell. It is thus possible to determine how many copies of any DNA sequence are present in a particular Deoxyribonucleic acid sample. The aforementioned technique can be used to search for related but nonidentical genes. To detect a gene of interest in an organism whose genome has not yet been sequenced, for case, a portion of a known factor can be used equally a probe (Figure 8-25).

Effigy 8-25

Different hybridization atmospheric condition permit less than perfect Dna matching. When only an identical match with a DNA probe is desired, the hybridization reaction is kept just a few degrees beneath the temperature at which a perfect Dna helix denatures in the (more...)

Alternatively, Dna probes can be used in hybridization reactions with RNA rather than Dna to find out whether a prison cell is expressing a given gene. In this case a DNA probe that contains office of the gene's sequence is hybridized with RNA purified from the prison cell in question to come across whether the RNA includes molecules matching the probe DNA and, if so, in what quantities. In somewhat more than elaborate procedures the DNA probe is treated with specific nucleases after the hybridization is consummate, to determine the exact regions of the Deoxyribonucleic acid probe that take paired with cellular RNA molecules. One tin can thereby make up one's mind the start and finish sites for RNA transcription, likewise as the precise boundaries of the intron and exon sequences in a gene (Figure 8-26).

Figure 8-26

The use of nucleic acid hybridization to decide the region of a cloned Dna fragment that is present in an mRNA molecule. The method shown requires a nuclease that cuts the DNA chain only where it is not base-paired to a complementary RNA chain. The (more than...)

Today, the positions of intron/exon boundaries are usually determined past sequencing the cDNA sequences that correspond the mRNAs expressed in a prison cell. Comparing this expressed sequence with the sequence of the whole gene reveals where the introns lie. Nosotros review subsequently how cDNAs are prepared from mRNAs.

We accept seen that genes are switched on and off as a cell encounters new signals in its environment. The hybridization of Dna probes to cellular RNAs allows i to make up one's mind whether or not a particular cistron is being transcribed; moreover, when the expression of a factor changes, one can determine whether the modify is due to transcriptional or posttranscriptional controls (come across Figure 7-87). These tests of gene expression were initially performed with one DNA probe at a time. DNA microarrays now permit the simultaneous monitoring of hundreds or thousands of genes at a time, as we talk over later. Hybridization methods are in such wide use in cell biology today that it is difficult to imagine how we could study cistron structure and expression without them.

Northern and Southern Blotting Facilitate Hybridization with Electrophoretically Separated Nucleic Acid Molecules

DNA probes are often used to notice, in a circuitous mixture of nucleic acids, only those molecules with sequences that are complementary to all or function of the probe. Gel electrophoresis can be used to fractionate the many unlike RNA or Deoxyribonucleic acid molecules in a crude mixture according to their size before the hybridization reaction is performed; if molecules of just one or a few sizes go labeled with the probe, one tin be certain that the hybridization was indeed specific. Moreover, the size information obtained can be invaluable in itself. An case illustrates this bespeak.

Suppose that one wishes to decide the nature of the defect in a mutant mouse that produces abnormally depression amounts of albumin, a protein that liver cells normally secrete into the blood in big amounts. Outset, i collects identical samples of liver tissue from mutant and normal mice (the latter serving equally controls) and disrupts the cells in a strong detergent to inactivate cellular nucleases that might otherwise degrade the nucleic acids. Next, ane separates the RNA and DNA from all of the other jail cell components: the proteins present are completely denatured and removed by repeated extractions with phenol—a potent organic solvent that is partly miscible with water; the nucleic acids, which remain in the aqueous phase, are and so precipitated with alcohol to divide them from the small molecules of the prison cell. So 1 separates the Deoxyribonucleic acid from the RNA by their different solubilities in alcohols and degrades any contaminating nucleic acrid of the unwanted blazon by treatment with a highly specific enzyme—either an RNase or a DNase. The mRNAs are typically separated from bulk RNA past retention on a chromatography column that specifically binds the poly-A tails of mRNAs.

To analyze the albumin-encoding mRNAs with a Dna probe, a technique called Northern blotting is used. First, the intact mRNA molecules purified from mutant and control liver cells are fractionated on the ground of their sizes into a series of bands by gel electrophoresis. And so, to make the RNA molecules accessible to DNA probes, a replica of the design of RNA bands on the gel is fabricated past transferring ("blotting") the fractionated RNA molecules onto a sail of nitrocellulose or nylon paper. The paper is and then incubated in a solution containing a labeled DNA probe whose sequence corresponds to office of the template strand that produces albumin mRNA. The RNA molecules that hybridize to the labeled Dna probe on the paper (because they are complementary to part of the normal albumin cistron sequence) are then located by detecting the spring probe by autoradiography or past chemical ways (Figure 8-27). The size of the RNA molecules in each band that binds the probe can be determined by reference to bands of RNA molecules of known sizes (RNA standards) that are electrophoresed next with the experimental sample. In this way one might find that liver cells from the mutant mice brand albumin RNA in normal amounts and of normal size; alternatively, albumin RNA of normal size might be detected in profoundly reduced amounts. Another possibility is that the mutant albumin RNA molecules might be abnormally brusque and therefore move unusually quickly through the gel; in this case the gel blot could be retested with a series of shorter DNA probes, each corresponding to small portions of the gene, to reveal which office of the normal RNA is missing.

Effigy 8-27

Detection of specific RNA or Dna molecules past gel-transfer hybridization. In this example, the DNA probe is detected by its radioactive decay. DNA probes detected by chemic or fluorescence methods are also widely used (see Figure 8-24). (A) A mixture of (more...)

An analogous gel-transfer hybridization method, chosen Southern blotting, analyzes DNA rather than RNA. Isolated Dna is beginning cut into readily separable fragments with restriction nucleases. The double-stranded fragments are then separated on the basis of size past gel electrophoresis, and those complementary to a DNA probe are identified by blotting and hybridization, as just described for RNA (encounter Figure 8-27). To characterize the construction of the albumin factor in the mutant mice, an albumin-specific Dna probe would be used to construct a detailed restriction map of the genome in the region of the albumin gene. From this map 1 could make up one's mind if the albumin gene has been rearranged in the defective animals—for example, past the deletion or the insertion of a curt DNA sequence; almost single base changes, even so, could non be detected in this way.

Hybridization Techniques Locate Specific Nucleic Acid Sequences in Cells or on Chromosomes

Nucleic acids, no less than other macromolecules, occupy precise positions in cells and tissues, and a nifty bargain of potential data is lost when these molecules are extracted by homogenization. For this reason, techniques take been developed in which nucleic acrid probes are used in much the aforementioned way as labeled antibodies to locate specific nucleic acid sequences in situ, a procedure chosen in situ hybridization. This process tin now be washed both for Dna in chromosomes and for RNA in cells. Labeled nucleic acid probes can be hybridized to chromosomes that have been exposed briefly to a very high pH to disrupt their Dna base of operations pairs. The chromosomal regions that bind the probe during the hybridization footstep are and so visualized. Originally, this technique was developed with highly radioactive Dna probes, which were detected by auto-radiography. The spatial resolution of the technique, however, can be greatly improved by labeling the Dna probes chemically (Figure eight-28) instead of radioactively, as described earlier.

In situ hybridization methods have also been developed that reveal the distribution of specific RNA molecules in cells in tissues. In this case the tissues are not exposed to a high pH, so the chromosomal DNA remains double-stranded and cannot bind the probe. Instead the tissue is gently fixed so that its RNA is retained in an exposed grade that can hybridize when the tissue is incubated with a complementary Deoxyribonucleic acid or RNA probe. In this manner the patterns of differential gene expression tin exist observed in tissues, and the location of specific RNAs can exist determined in cells (Figure viii-29). In the Drosophila embryo, for case, such patterns have provided new insights into the mechanisms that create distinctions between cells in different positions during development (described in Chapter 21).

Figure 8-29

(A) Expression blueprint of deltaC in the early on zebrafish embryo. This gene codes for a ligand in the Notch signaling pathway (discussed in Chapter 15), and the pattern shown here reflects its function in the development of somites—the future segments (more than...)

Genes Tin Be Cloned from a Dna Library

Any Dna fragment that contains a gene of interest can be cloned. In cell biology, the term Deoxyribonucleic acid cloning is used in two senses. In one sense it literally refers to the human action of making many identical copies of a Deoxyribonucleic acid molecule—the amplification of a particular DNA sequence. However, the term is as well used to describe the isolation of a item stretch of DNA (often a detail gene) from the rest of a cell'due south Dna, considering this isolation is greatly facilitated by making many identical copies of the Dna of interest.

Dna cloning in its most full general sense tin be accomplished in several ways. The simplest involves inserting a particular fragment of Dna into the purified DNA genome of a self-replicating genetic element—generally a virus or a plasmid. A DNA fragment containing a human gene, for example, can exist joined in a test tube to the chromosome of a bacterial virus, and the new recombinant DNA molecule can then be introduced into a bacterial cell. Starting with merely one such recombinant Deoxyribonucleic acid molecule that infects a unmarried cell, the normal replication mechanisms of the virus can produce more than ten¹² identical virus DNA molecules in less than a day, thereby amplifying the amount of the inserted human being DNA fragment by the same factor. A virus or plasmid used in this style is known as a cloning vector, and the DNA propagated by insertion into it is said to take been cloned.

To isolate a specific gene, i frequently begins by constructing a DNA library—a comprehensive drove of cloned DNA fragments from a jail cell, tissue, or organism. This library includes (one hopes) at to the lowest degree one fragment that contains the gene of interest. Libraries can exist constructed with either a virus or a plasmid vector and are by and large housed in a population of bacterial cells. The principles underlying the methods used for cloning genes are the aforementioned for either type of cloning vector, although the details may differ. Today almost cloning is performed with plasmid vectors.

The plasmid vectors well-nigh widely used for gene cloning are small circular molecules of double-stranded Dna derived from larger plasmids that occur naturally in bacterial cells. They more often than not account for only a small-scale fraction of the total host bacterial cell DNA, but they can easily be separated attributable to their small-scale size from chromosomal Deoxyribonucleic acid molecules, which are large and precipitate as a pellet upon centrifugation. For use every bit cloning vectors, the purified plasmid DNA circles are first cutting with a restriction nuclease to create linear Dna molecules. The cellular DNA to be used in constructing the library is cut with the same restriction nuclease, and the resulting restriction fragments (including those containing the factor to be cloned) are so added to the cut plasmids and annealed via their cohesive ends to form recombinant DNA circles. These recombinant molecules containing foreign DNA inserts are then covalently sealed with the enzyme DNA ligase (Figure viii-30).

Figure 8-xxx

The insertion of a DNA fragment into a bacterial plasmid with the enzyme Dna ligase. The plasmid is cut open with a restriction nuclease (in this case one that produces cohesive ends) and is mixed with the Dna fragment to be cloned (which has been prepared (more...)

In the next step in preparing the library, the recombinant Dna circles are introduced into bacterial cells that accept been made transiently permeable to DNA; such cells are said to exist transfected with the plasmids. Equally these cells abound and split, doubling in number every 30 minutes, the recombinant plasmids as well replicate to produce an enormous number of copies of DNA circles containing the foreign Dna (Figure eight-31). Many bacterial plasmids acquit genes for antibiotic resistance, a holding that can be exploited to select those cells that have been successfully transfected; if the leaner are grown in the presence of the antibiotic, only cells containing plasmids will survive. Each original bacterial cell that was initially transfected contains, in general, a dissimilar foreign DNA insert; this insert is inherited past all of the progeny cells of that bacterium, which together form a small colony in a culture dish.

Effigy viii-31

Purification and amplification of a specific Deoxyribonucleic acid sequence past DNA cloning in a bacterium. To produce many copies of a particular DNA sequence, the fragment is first inserted into a plasmid vector, equally shown in Figure 8-30. The resulting recombinant plasmid (more than...)

For many years, plasmids were used to clone fragments of Deoxyribonucleic acid of ane,000 to xxx,000 nucleotide pairs. Larger DNA fragments are more difficult to handle and were harder to clone. Then researchers began to use yeast artificial chromosomes (YACs), which could handle very large pieces of Deoxyribonucleic acid (Figure eight-32). Today, new plasmid vectors based on the naturally occurring F plasmid of E. coli are used to clone Dna fragments of 300,000 to i one thousand thousand nucleotide pairs. Different smaller bacterial plasmids, the F plasmid—and its derivative, the bacterial artificial chromosome (BAC)—is nowadays in but one or two copies per Due east. coli jail cell. The fact that BACs are kept in such depression numbers in bacterial cells may contribute to their ability to maintain large cloned Deoxyribonucleic acid sequences stably: with only a few BACs present, information technology is less likely that the cloned DNA fragments will go scrambled due to recombination with sequences carried on other copies of the plasmid. Because of their stability, power to have big DNA inserts, and ease of handling, BACs are now the preferred vector for building DNA libraries of complex organisms—including those representing the human and mouse genomes.

Figure 8-32

The making of a yeast bogus chromosome (YAC). A YAC vector allows the cloning of very big DNA molecules. TEL, CEN, and ORI are the telomere, centromere, and origin of replication sequences, respectively, for the yeast Saccharomyces cerevisiae. (more...)

Two Types of DNA Libraries Serve Different Purposes

Cleaving the entire genome of a cell with a specific restriction nuclease and cloning each fragment equally just described is sometimes called the "shotgun" approach to factor cloning. This technique tin produce a very large number of DNA fragments—on the order of a 1000000 for a mammalian genome—which will generate millions of different colonies of transfected bacterial cells. (When working with BACs rather than typical plasmids, larger fragments can be inserted, so fewer transfected bacterial cells are required to comprehend the genome.) Each of these colonies is equanimous of a clone of cells derived from a unmarried ancestor cell, and therefore harbors many copies of a particular stretch of the fragmented genome (Figure 8-33). Such a plasmid is said to incorporate a genomic Dna clone, and the entire collection of plasmids is called a genomic Dna library. Simply considering the genomic Dna is cut into fragments at random, only some fragments contain genes. Many of the genomic DNA clones obtained from the DNA of a higher eucaryotic jail cell contain only noncoding DNA, which, every bit we discussed in Affiliate four, makes upward near of the DNA in such genomes.

Figure 8-33

Construction of a human genomic Deoxyribonucleic acid library. A genomic library is usually stored as a set of bacteria, each carrying a different fragment of human Dna. For simplicity, cloning of just a few representative fragments (colored) is shown. In reality, all (more...)

An culling strategy is to begin the cloning procedure by selecting only those DNA sequences that are transcribed into mRNA and thus are presumed to represent to poly peptide-encoding genes. This is done by extracting the mRNA (or a purified subfraction of the mRNA) from cells and then making a complementary Deoxyribonucleic acid (cDNA) copy of each mRNA molecule present; this reaction is catalyzed past the reverse transcriptase enzyme of retroviruses, which synthesizes a DNA chain on an RNA template. The single-stranded DNA molecules synthesized by the reverse transcriptase are converted into double-stranded DNA molecules by DNA polymerase, and these molecules are inserted into a plasmid or virus vector and cloned (Figure 8-34). Each clone obtained in this way is called a cDNA clone, and the unabridged collection of clones derived from i mRNA preparation constitutes a cDNA library.

Figure 8-34

The synthesis of cDNA. Total mRNA is extracted from a particular tissue, and Dna copies (cDNA) of the mRNA molecules are produced by the enzyme reverse transcriptase (see p. 289). For simplicity, the copying of just i of these mRNAs into cDNA is illustrated. (more...)

There are important differences betwixt genomic Dna clones and cDNA clones, as illustrated in Effigy eight-35. Genomic clones represent a random sample of all of the Dna sequences in an organism and, with very rare exceptions, are the same regardless of the cell type used to prepare them. Past contrast, cDNA clones contain only those regions of the genome that accept been transcribed into mRNA. Because the cells of different tissues produce distinct sets of mRNA molecules, a distinct cDNA library is obtained for each type of cell used to prepare the library.

Effigy 8-35

The differences between cDNA clones and genomic Deoxyribonucleic acid clones derived from the same region of Deoxyribonucleic acid. In this instance factor A is infrequently transcribed, whereas gene B is ofttimes transcribed, and both genes contain introns (green). In the genomic DNA library, (more...)

cDNA Clones Contain Uninterrupted Coding Sequences

The employ of a cDNA library for gene cloning has several advantages. First, some proteins are produced in very large quantities by specialized cells. In this instance, the mRNA encoding the protein is likely to exist produced in such large quantities that a cDNA library prepared from the cells is highly enriched for the cDNA molecules encoding the protein, greatly reducing the problem of identifying the desired clone in the library (run into Figure 8-35). Hemoglobin, for example, is made in large amounts by developing erythrocytes (red blood cells); for this reason the globin genes were among the first to be cloned.

By far the nearly important advantage of cDNA clones is that they contain the uninterrupted coding sequence of a gene. Every bit we accept seen, eucaryotic genes usually consist of brusque coding sequences of DNA (exons) separated by much longer noncoding sequences (introns); the product of mRNA entails the removal of the noncoding sequences from the initial RNA transcript and the splicing together of the coding sequences. Neither bacterial nor yeast cells will brand these modifications to the RNA produced from a gene of a college eucaryotic jail cell. Thus, when the aim of the cloning is either to deduce the amino acid sequence of the poly peptide from the Dna sequence or to produce the protein in bulk by expressing the cloned cistron in a bacterial or yeast prison cell, it is much preferable to outset with cDNA.

Genomic and cDNA libraries are inexhaustible resource that are widely shared amongst investigators. Today, many such libraries are also bachelor from commercial sources.

Isolated Dna Fragments Can Be Rapidly Sequenced

In the tardily 1970s methods were developed that allowed the nucleotide sequence of any purified DNA fragment to exist determined only and chop-chop. They take fabricated it possible to decide the complete DNA sequences of tens of thousands of genes, and many organisms have had their Dna genomes fully sequenced (see Table 1-i, p. twenty). The book of Dna sequence information is now so large (many tens of billions of nucleotides) that powerful computers must exist used to store and analyze information technology.

Large volume DNA sequencing was made possible through the development in the mid-1970s of the dideoxy method for sequencing DNA, which is based on in vitro Deoxyribonucleic acid synthesis performed in the presence of chain-terminating dideoxyribonucleoside triphosphates (Effigy viii-36).

Figure 8-36

The enzymatic—or dideoxy—method of sequencing Deoxyribonucleic acid. (A) This method relies on the use of dideoxyribonucleoside triphosphates, derivatives of the normal deoxyribonucleoside triphosphates that lack the iii′ hydroxyl grouping. (B) Purified (more...)

Although the same bones method is even so used today, many improvements take been made. Deoxyribonucleic acid sequencing is at present completely automated: robotic devices mix the reagents and then load, run, and read the order of the nucleotide bases from the gel. This is facilitated by using chain-terminating nucleotides that are each labeled with a unlike colored fluorescent dye; in this example, all four synthesis reactions tin can be performed in the same tube, and the products tin can be separated in a single lane of a gel. A detector positioned near the bottom of the gel reads and records the color of the fluorescent label on each band as it passes through a laser beam (Figure viii-37). A computer and then reads and stores this nucleotide sequence.

Figure 8-37

Automatic DNA sequencing. Shown here is a tiny office of the data from an automated Dna-sequencing run as it appears on the computer screen. Each colored height represents a nucleotide in the DNA sequence—a articulate stretch of nucleotide sequence can (more...)

Nucleotide Sequences Are Used to Predict the Amino Acid Sequences of Proteins

At present that Dna sequencing is so rapid and reliable, it has become the preferred method for determining, indirectly, the amino acid sequences of most proteins. Given a nucleotide sequence that encodes a protein, the procedure is quite straightforward. Although in principle there are six unlike reading frames in which a DNA sequence can exist translated into protein (three on each strand), the correct one is by and large recognizable as the merely i lacking frequent stop codons (Figure eight-38). Equally nosotros saw when nosotros discussed the genetic code in Affiliate 6, a random sequence of nucleotides, read in frame, volition encode a stop signal for protein synthesis about once every 20 amino acids. Those nucleotide sequences that encode a stretch of amino acids much longer than this are candidates for presumptive exons, and they can be translated (by computer) into amino acid sequences and checked confronting databases for similarities to known proteins from other organisms. If necessary, a limited amount of amino acrid sequence tin can and then be determined from the purified protein to confirm the sequence predicted from the DNA.

Figure viii-38

Finding the regions in a DNA sequence that encode a protein. (A) Any region of the Deoxyribonucleic acid sequence tin, in principle, code for 6 different amino acid sequences, considering whatsoever one of three different reading frames tin be used to interpret the nucleotide sequence (more...)

The problem comes, however, in determining which nucleotide sequences—within a whole genome sequence—stand for genes that encode proteins. Identifying genes is easiest when the DNA sequence is from a bacterial or archeal chromosome, which lacks introns, or from a cDNA clone. The location of genes in these nucleotide sequences tin be predicted by examining the DNA for certain distinctive features (discussed in Affiliate 6). Briefly these genes that encode proteins are identified by searching the nucleotide sequence for open up reading frames (ORFs) that begin with an initiation codon, usually ATG, and end with a termination codon, TAA, TAG, or TGA. To minimize errors, computers used to search for ORFs are often directed to count as genes only those sequences that are longer than, say, 100 codons in length.

For more complex genomes, such as those of eucaryotes, the procedure is complicated by the presence of big introns embedded within the coding portion of genes. In many multicellular organisms, including humans, the average exon is only 150 nucleotides long. Thus in eucaryotes, one must besides search for other features that point the presence of a factor, for example, sequences that signal an intron/exon boundary or distinctive upstream regulatory regions.

A 2d major arroyo to identifying the coding regions in chromosomes is through the characterization of the nucleotide sequences of the detectable mRNAs (in the class of cDNAs). The mRNAs (and the cDNAs produced from them) lack introns, regulatory DNA sequences, and the nonessential "spacer" Dna that lies betwixt genes. It is therefore useful to sequence large numbers of cDNAs to produce a very large collection (chosen a database) of the coding sequences of an organism. These sequences are then readily used to distinguish the exons from the introns in the long chromosomal Dna sequences that correspond to genes.

Finally, nucleotide sequences that are conserved betwixt closely related organisms normally encode proteins. Comparison of these conserved sequences in unlike species tin also provide insight into the role of a particular poly peptide or gene, every bit nosotros run into later in the chapter.

The Genomes of Many Organisms Accept Been Fully Sequenced

Owing in large part to the automation of DNA sequencing, the genomes of many organisms accept been fully sequenced; these include institute chloroplasts and animal mitochondria, large numbers of bacteria and archea, and many of the model organisms that are studied routinely in the laboratory, including several yeasts, a nematode worm, the fruit fly Drosophila, the model establish Arabidopsis, the mouse, and, terminal merely not least, humans. Researchers have also deduced the complete Deoxyribonucleic acid sequences for a wide variety of human pathogens. These include the bacteria that cause cholera, tuberculosis, syphilis, gonorrhea, Lyme disease, and stomach ulcers, as well as hundreds of viruses—including smallpox virus and Epstein-Barr virus (which causes infectious mononucleosis). Examination of the genomes of these pathogens should provide clues about what makes them virulent, and will also point the style to new and more than effective treatments.

Haemophilus influenzae (a bacterium that can cause ear infections or meningitis in children) was the first organism to have its consummate genome sequence—all one.8 meg nucleotides—adamant by the shotgun sequencing method, the almost common strategy used today. In the shotgun method, long sequences of Dna are broken apart randomly into many shorter fragments. Each fragment is then sequenced and a computer is used to order these pieces into a whole chromosome or genome, using sequence overlap to guide the associates. The shotgun method is the technique of choice for sequencing pocket-sized genomes. Although larger, more repetitive genome sequences are more catchy to get together, the shotgun method has been useful for sequencing the genomes of Drosophila melanogaster, mouse, and human being.

With new sequences appearing at a steadily accelerating pace in the scientific literature, comparison of the complete genome sequences of unlike organisms allows us to trace the evolutionary relationships amidst genes and organisms, and to discover genes and predict their functions. Assigning functions to genes oftentimes involves comparison their sequences with related sequences from model organisms that accept been well characterized in the laboratory, such as the bacterium E. coli, the yeasts Southward. cerevisiae and Due south. pombe, the nematode worm C. elegans, and the fruit fly Drosophila (discussed in Affiliate 1).

Although the organisms whose genomes accept been sequenced share many cellular pathways and possess many proteins that are homologous in their amino acrid sequences or structure, the functions of a very large number of newly identified proteins remain unknown. Some xv–40% of the proteins encoded by these sequenced genomes practise not resemble any other protein that has been characterized functionally. This ascertainment underscores one of the limitations of the emerging field of genomics: although comparative analysis of genomes reveals a bully deal of information about the relationships between genes and organisms, it frequently does not provide firsthand information about how these genes part, or what roles they accept in the physiology of an organism. Comparing of the total gene complement of several thermophilic bacteria, for example, does not reveal why these bacteria thrive at temperatures exceeding 70°C. And examination of the genome of the incredibly radioresistant bacterium Deinococcus radiodurans does not explicate how this organism tin survive a nail of radiation that can shatter glass. Further biochemical and genetic studies, like those described in the final sections of this chapter, are required to determine how genes part in the context of living organisms.

Selected Deoxyribonucleic acid Segments Can Be Cloned in a Examination Tube past a Polymerase Chain Reaction

At present that so many genome sequences are available, genes can be cloned directly without the need to construct Deoxyribonucleic acid libraries offset. A technique called the polymerase concatenation reaction (PCR) makes this rapid cloning possible. PCR allows the Dna from a selected region of a genome to be amplified a billionfold, effectively "purifying" this Deoxyribonucleic acid abroad from the residual of the genome.

Two sets of Deoxyribonucleic acid oligonucleotides, chosen to flank the desired nucleotide sequence of the gene, are synthesized by chemical methods. These oligonucleotides are then used to prime DNA synthesis on unmarried strands generated by heating the Dna from the entire genome. The newly synthesized Deoxyribonucleic acid is produced in a reaction catalyzed in vitro by a purified Dna polymerase, and the primers remain at the 5′ ends of the terminal Deoxyribonucleic acid fragments that are made (Figure 8-39A).

Effigy 8-39

Amplification of DNA using the PCR technique. Knowledge of the Dna sequence to exist amplified is used to design two synthetic Deoxyribonucleic acid oligonucleotides, each complementary to the sequence on i strand of the DNA double helix at contrary ends of the region to (more than...)

Cypher special is produced in the first bike of Dna synthesis; the ability of the PCR method is revealed only subsequently repeated rounds of DNA synthesis. Every bicycle doubles the corporeality of DNA synthesized in the previous cycle. Considering each cycle requires a brief rut handling to separate the two strands of the template DNA double helix, the technique requires the use of a special DNA polymerase, isolated from a thermophilic bacterium, that is stable at much higher temperatures than normal, so that it is not denatured by the repeated heat treatments. With each round of Dna synthesis, the newly generated fragments serve as templates in their plough, and within a few cycles the predominant product is a single species of DNA fragment whose length corresponds to the altitude betwixt the two original primers (run across Figure viii-39B).

In do, 20–30 cycles of reaction are required for effective Dna amplification, with the products of each bike serving every bit the Deoxyribonucleic acid templates for the next—hence the term polymerase "chain reaction." A unmarried cycle requires only well-nigh v minutes, and the entire procedure tin exist easily automatic. PCR thereby makes possible the "cell-free molecular cloning" of a Deoxyribonucleic acid fragment in a few hours, compared with the several days required for standard cloning procedures. This technique is now used routinely to clone DNA from genes of interest directly—starting either from genomic DNA or from mRNA isolated from cells (Figure 8-40).

Figure 8-40

Utilize of PCR to obtain a genomic or cDNA clone. (A) To obtain a genomic clone past using PCR, chromosomal Deoxyribonucleic acid is first purified from cells. PCR primers that flank the stretch of Deoxyribonucleic acid to exist cloned are added, and many cycles of the reaction are completed (encounter (more...)

The PCR method is extremely sensitive; it can notice a single Deoxyribonucleic acid molecule in a sample. Trace amounts of RNA can be analyzed in the same way by offset transcribing them into DNA with opposite transcriptase. The PCR cloning technique has largely replaced Southern blotting for the diagnosis of genetic diseases and for the detection of low levels of viral infection. It besides has smashing promise in forensic medicine every bit a means of analyzing minute traces of blood or other tissues—fifty-fifty as little as a single cell—and identifying the person from whom they came by his or her genetic "fingerprint" (Figure 8-41).

Figure 8-41

How PCR is used in forensic scientific discipline. (A) The Deoxyribonucleic acid sequences that create the variability used in this analysis contain runs of short, repeated sequences, such as CACACA . . . , which are institute in diverse positions (loci) in the human genome. The number (more...)

Cellular Proteins Can Exist Fabricated in Big Amounts Through the Apply of Expression Vectors

Xv years agone, the only proteins in a cell that could be studied hands were the relatively abundant ones. Starting with several hundred grams of cells, a major protein—one that constitutes ane% or more than of the total cellular protein—can be purified by sequential chromatography steps to yield perhaps 0.i g (100 mg) of pure poly peptide. This corporeality was sufficient for conventional amino acid sequencing, for detailed analysis of biochemical activities, and for the production of antibodies, which could then be used to localize the poly peptide in the prison cell. Moreover, if suitable crystals could be grown (frequently a difficult job), the three-dimensional structure of the protein could be determined by x-ray diffraction techniques, as we will hash out later. The structure and function of many arable proteins—including hemoglobin, trypsin, immunoglobulin, and lysozyme—were analyzed in this style.

The vast majority of the thousands of dissimilar proteins in a eucaryotic prison cell, however, including many with crucially important functions, are nowadays in very small amounts. For most of them it is extremely hard, if not impossible, to obtain more than a few micrograms of pure material. One of the nearly important contributions of DNA cloning and genetic engineering to cell biology is that they have made information technology possible to produce any of the prison cell's proteins in nearly unlimited amounts.

Large amounts of a desired protein are produced in living cells by using expression vectors (Figure eight-42). These are mostly plasmids that have been designed to produce a large corporeality of a stable mRNA that can be efficiently translated into protein in the transfected bacterial, yeast, insect, or mammalian jail cell. To foreclose the high level of the strange protein from interfering with the transfected cell's growth, the expression vector is often designed so that the synthesis of the foreign mRNA and protein tin can exist delayed until shortly before the cells are harvested (Figure eight-43).

Figure 8-42

Production of big amounts of a protein from a protein-coding DNA sequence cloned into an expression vector and introduced into cells. A plasmid vector has been engineered to incorporate a highly agile promoter, which causes unusually large amounts of mRNA (more than...)

Effigy 8-43

Production of large amounts of a protein by using a plasmid expression vector. In this example, bacterial cells have been transfected with the coding sequence for an enzyme, Deoxyribonucleic acid helicase; transcription from this coding sequence is under the command of (more...)

Because the desired protein fabricated from an expression vector is produced inside a jail cell, it must be purified away from the host cell proteins by chromatography following prison cell lysis; merely considering it is such a plentiful species in the jail cell lysate (often ane–10% of the total cell poly peptide), the purification is usually easy to reach in simply a few steps. Many expression vectors accept been designed to add a molecular tag—a cluster of histidine residues or a small marker protein—to the expressed protein to make possible like shooting fish in a barrel purification by affinity chromatography, equally discussed previously (see pp. 483–484). A variety of expression vectors are available, each engineered to function in the type of cell in which the poly peptide is to be fabricated. In this way cells can be induced to make vast quantities of medically useful proteins—such as human being insulin and growth hormone, interferon, and viral antigens for vaccines. More more often than not, these methods make it possible to produce every poly peptide—even those that may exist present in but a few copies per cell—in large plenty amounts to be used in the kinds of detailed structural and functional studies that we talk over in the next department (Effigy eight-44).

Figure 8-44

Knowledge of the molecular biological science of cells makes it possible to experimentally move from factor to protein and from poly peptide to gene. A pocket-size quantity of a purified poly peptide is used to obtain a partial amino acid sequence. This provides sequence information (more...)

DNA technology can also be used to produce large amounts of any RNA molecule whose gene has been isolated. Studies of RNA splicing, poly peptide synthesis, and RNA-based enzymes, for instance, ar greatly facilitated by the availability of pure RNA molecules. Most RNAs are present in but tiny quantities in cells, and they are very difficult to purify abroad from other cellular components—specially from the many thousands of other RNAs present in the prison cell. Only any RNA of interest tin can exist synthesized efficiently in vitro by transcription of its Dna sequence with a highly efficient viral RNA polymerase. The unmarried species of RNA produced is then easily purified abroad from the Dna template and the RNA polymerase.

Summary

Deoxyribonucleic acid cloning allows a copy of whatever specific office of a DNA or RNA sequence to be selected from the millions of other sequences in a jail cell and produced in unlimited amounts in pure form. DNA sequences can be amplified after cutting chromosomal Deoxyribonucleic acid with a restriction nuclease and inserting the resulting DNA fragments into the chromosome of a self-replicating genetic element. Plasmid vectors are more often than not used and the resulting "genomic DNA library" is housed in millions of bacterial cells, each conveying a different cloned Deoxyribonucleic acid fragment. Private cells that are allowed to proliferate produce large amounts of a single cloned DNA fragment from this library. As an alternative, the polymerase chain reaction (PCR) allows Dna cloning to be performed directly with a purified, thermostable DNA polymerase—providing that the Deoxyribonucleic acid sequence of interest is already known.

The procedures used to obtain Dna clones that correspond in sequence to mRNA molecules are the same except that a DNA copy of the mRNA sequence, chosen cDNA, is get-go made. Different genomic Deoxyribonucleic acid clones, cDNA clones lack intron sequences, making them the clones of choice for analyzing the poly peptide product of a gene.

Nucleic acid hybridization reactions provide a sensitive means of detecting a gene or whatever other nucleotide sequence of choice. Under stringent hybridization conditions (a combination of solvent and temperature where a perfect double helix is barely stable), two strands tin pair to course a "hybrid" helix only if their nucleotide sequences are well-nigh perfectly complementary. The enormous specificity of this hybridization reaction allows any single-stranded sequence of nucleotides to be labeled with a radioisotope or chemical and used every bit a probe to observe a complementary partner strand, fifty-fifty in a cell or cell extract that contains millions of different DNA and RNA sequences. Probes of this type are widely used to detect the nucleic acids corresponding to specific genes, both to facilitate their purification and label and to localize them in cells, tissues, and organisms.

The nucleotide sequence of purified Deoxyribonucleic acid fragments can exist determined speedily and only past using highly automated techniques based on the dideoxy method for sequencing DNA. This technique has made it possible to decide the complete DNA sequences of tens of thousands of genes and to completely sequence the genomes of many organisms. Comparing of the genome sequences of different organisms allows the states to trace the evolutionary relationships amidst genes and organisms, and it has proved valuable for discovering new genes and predicting their part.

Taken together, these techniques have made information technology possible to identify, isolate, and sequence genes from whatsoever organism of interest. Related technologies allow scientists to produce the protein products of these genes in the large quantities needed for detailed analyses of their structure and office, as well as for medical purposes.

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Source: https://www.ncbi.nlm.nih.gov/books/NBK26837/

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