Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

EcoRI

Digest with

HindIII and BamHI

FIGURE 13.6 ● Directional cloning. DNA molecules whose ends have different overhangs can be used to form chimeric constructs in which the foreign DNA can enter the plasmid in only one orientation. The foreign DNA is digested with two different restriction enzymes (HindIII and BamHI), and the plasmid is digested with the same two enzymes. Note that pUC19 has a polylinker or universal cloning site (see Figure 13.5b); pUC stands for universal cloning plasmid.

pBR322 (4363 bases)

AvaI

SalI

ori

Pvu

II

1A plasmid with genes for ampicillin resistance (ampr) and tetracycline resistance (tetr).

A BamHI restriction site is located within the tetr gene.

Tetracycline-containing medium

8Only tetr colonies appear; tets colonies can be recovered from ampr plate by comparing two plates.

● A typical bacterial transformation experiment. Here the plasmid pBR322 is the cloning vector. (1) Cleavage of pBR322 with restriction enzyme BamH1, followed by (2) annealing and ligation of inserts generated by BamH1 cleavage of some foreign DNA, (3) creates a chimeric plasmid. (4) The chimeric plasmid is then used to transform Ca2 -treated heat-shocked E. coli cells, and the bacterial sample is plated on a petri plate. (5) Following incubation of the petri plate overnight at 37°C, (6) colonies of ampr bacteria are evident. (7) Replica plating of these bacteria on plates of tetracycline-contain- ing media (8) reveals which colonies are tetr and which are tetracycline sensitive (tets). Only the tets colonies possess plasmids with foreign DNA inserts.

cation of exogenous DNA by a recipient cell; see Chapter 29). The bacterial cells were rendered somewhat permeable to DNA by Ca2 treatment and a brief 42°C heat shock. Although less than 0.1% of the Ca2 -treated bacteria became competent for transformation following such treatment, transformed bacteria could be selected by their resistance to certain antibiotics (Figure 13.7). Consequently, the chimeric plasmids must have been biologically functional in at least two aspects: they replicated stably within their hosts and they expressed the drug resistance markers they carried.

In general, plasmids used as cloning vectors are engineered to be small, 2.5 kbp to about 10 kbp in size, so that the size of the insert DNA can be maximized. These plasmids have only a single origin of replication, so the time necessary for complete replication depends on the size of the plasmid. Under selective pressure in a growing culture of bacteria, overly large plasmids are prone to delete any nonessential “genes,” such as any foreign inserts. Such deletion would thwart the purpose of most cloning experiments. The useful upper limit on cloned inserts in plasmids is about 10 kbp. Many eukaryotic genes exceed this size.

Bacteriophage as a Cloning Vector

The genome of bacteriophage (lambda) (Figure 13.8) is a 48.5-kbp linear DNA molecule that is packaged into the head of the bacteriophage. The middle one-third of this genome is not essential to phage infection, so phage DNA has been engineered so that foreign DNA molecules up to 16 kbp can be inserted into this region for cloning purposes. In vitro packaging systems are then used to package the chimeric DNA into phage heads which, when assembled with phage tails, form infective phage particles. Bacteria infected with these recombinant phage produce large numbers of phage progeny before they lyse, and large amounts of recombinant DNA can be easily purified from the lysate.

C o s m i d s

The DNA incorporated into phage heads by bacteriophage packaging systems must satisfy only a few criteria. It must possess a 14-bp sequence known as cos (which stands for cohesive end site) at each of its ends, and these cos sequences must be separated by no fewer than 36 kbp and no more than 51 kbp of DNA. Essentially any DNA satisfying these minimal requirements will be packaged and assembled into an infective phage particle. Other cloning features such as an ori, selectable markers, and a polylinker are joined to the cos sequence so that the cloned DNA can be propagated and selected in host cells. These features have been achieved by placing cos sequences on either side of cloning sites in plasmids to create cosmid vectors that are capable of carrying DNA inserts about 40 kbp in size (Figure 13.9). Because cosmids lack essential phage genes, they reproduce in host bacteria as plasmids.

Shuttle Vectors

Shuttle vectors are plasmids capable of propagating and transferring (“shuttling”) genes between two different organisms, one of which is typically a prokaryote (E. coli) and the other a eukaryote (for example, yeast). Shuttle vectors must have unique origins of replication for each cell type as well as different markers for selection of transformed host cells harboring the vector (Figure 13.10). Shuttle vectors have the advantage that eukaryotic genes can be cloned in bacterial hosts, yet the expression of these genes can be analyzed in appropriate eukaryotic backgrounds.

FIGURE 13.8 ● Electron micrograph of bacteriophage . (Robley C. Williams, University of California/BPS)

Combinatorial Libraries

Specific recognition and binding of other molecules is a defining characteristic of any protein or nucleic acid. Often, target ligands of a particular protein are unknown, or, in other instances, a unique ligand for a known protein may be sought in the hope of blocking the activity of the protein or otherwise perturbing its function. Combinatorial libraries are the products of emerging strategies to facilitate the identification and characterization of possible ligands for a protein. These strategies are also applicable to the study of nucleic acids. Unlike genomic libraries, combinatorial libraries consist of synthetic oligomers. Arrays of synthetic oligonucleotides printed as tiny dots on miniature solid supports are known as DNA chips. Specifically, combinatorial libraries contain very large numbers of chemically synthesized molecules (such as peptides or oligonucleotides) with randomized sequences or structures. Such libraries are designed and constructed with the hope that one molecule among a vast number will be recognized as a ligand by the protein (or nucleic acid) of interest. If so, perhaps that molecule will be useful in a pharmaceutical application, for instance as a drug to treat a disease involving the protein to which it binds.

An example of this strategy is the preparation of a synthetic combinatorial library of hexapeptides. The maximum number of sequence combinations for hexapeptides is 206 or 64,000,000. One approach to simplify preparation and screening possibilities for such a library is to specify the first two amino acids in the hexa-

peptide while the next four are randomly chosen. In this approach, 400 libraries (202) are synthesized, each of which is unique in terms of the amino acids at positions 1 and 2 but random at the other four positions (as in AAXXXX, ACXXXX, ADXXXX, etc.) so that each of the 400 libraries contains 204 or 160,000 different sequence combinations. Screening these libraries with the protein of interest reveals which of the 400 libraries contains a ligand with high affinity. This library is then systematically expanded by specifying the first 3 amino acids (knowing from the chosen 1-of-400 libraries which amino acids are best as the first 2); only 20 synthetic libraries (each containing 203 or 8000 hexapeptides) are made here (one for each third-position possibility, the remaining three positions being randomized). Selection for ligand binding, again with the protein of interest, reveals the best of these 20, and this particular library is then varied systematically at the fourth position, creating 20 more libraries (each containing 202 or 400 hexapeptides). This cycle of synthesis, screening, and selection is repeated until all six positions in the hexapeptide are optimized to create the best ligand for the protein. A variation on this basic strategy using synthetic oligonucleotides rather than peptides identified a unique 15-mer (sequence GGTTGGTGTGGTTGG) with high affinity (KD 2.7 nM) toward thrombin, a serine protease in the blood coagulation pathway. Thrombin is a major target for the pharmacological prevention of clot formation in coronary thrombosis.

● Screening a genomic library by colony hybridization (or plaque hybridization). Host bacteria transformed with a plasmid-based genomic library or infected with a bacteriophage-based genomic library are plated on a petri plate and incubated overnight to allow bacterial colonies (or phage plaques) to form. A replica of the bacterial colonies (or plaques) is then obtained by overlaying the plate with a nitrocellulose disc (1). Nitrocellulose strongly binds nucleic acids; single-stranded nucleic acids are bound more tightly than double-stranded nucleic acids. (Nylon membranes with similar nucleic acid – and protein-binding properties are also used.) Once the nitrocellulose disc has taken up an impression of the bacterial colonies (or plaques), it is removed and the petri plate is set aside and saved. The disc is treated with 2 M NaOH, neutralized, and dried (2). NaOH both lyses any bacteria (or phage particles) and dissociates the DNA strands. When the disc is dried, the DNA strands become immobilized on the filter. The dried disc is placed in a sealable plastic bag, and a solution containing heat-denatured (single-stranded), labeled probe is added (3). The bag is incubated to allow annealing of the probe DNA to any target DNA sequences that might be present on the nitrocellulose. The filter is then washed, dried, and placed on a piece of X-ray film to obtain an autoradiogram (4). The position of any spots on the X-ray film reveals where the labeled probe has hybridized with target DNA (5). The location of these spots can be used to recover the genomic clone from the bacteria (or plaques) on the original petri plate.

13.2 ● DNA Libraries 407

Master plate of bacteria colonies (or phage plaques)

1

Replicate onto nitrocellulose disc

2

Treat with NaOH; neutralize, dry

Denatured DNA bound to nitrocellulose

sequence within the 3 106 kbp human genome, N would equal almost 1.4 million if the cloned fragments averaged 10 kbp in size. The need for cloning vectors capable of carrying very large DNA inserts becomes obvious from these numbers.

Screening Libraries

A common method of screening plasmid-based genomic libraries is to carry out a colony hybridization experiment. The protocol is similar for phage-based libraries except that bacteriophage plaques, not bacterial colonies, are screened. In a typical experiment, host bacteria containing either a plasmidbased or bacteriophage-based library are plated out on a petri dish and allowed to grow overnight to form colonies (or in the case of phage libraries, plaques) (Figure 13.11). A replica of the bacterial colonies (or plaques) is then obtained by overlaying the plate with a nitrocellulose disc. The disc is removed, treated with alkali to dissociate bound DNA duplexes into single-stranded DNA, dried, and placed in a sealed bag with labeled probe (see the box on Southern blotting). If the probe DNA is duplex DNA, it must be denatured by heating at 70°C. The probe and target DNA complementary sequences must be in a singlestranded form if they are to hybridize with one another. Any DNA sequences complementary to probe DNA will be revealed by autoradiography of the nitrocellulose disc. Bacterial colonies (phage plaques) containing clones bearing target DNA are identified on the film and can be recovered from the master plate.

Probes for Southern Hybridization

Clearly, specific probes are essential reagents if the goal is to identify a particular gene against a background of innumerable DNA sequences. Usually, the probes that are used to screen libraries are nucleotide sequences that are complementary to some part of the target gene. To make useful probes requires some information about the gene’s nucleotide sequence. Sometimes such information is available. Alternatively, if the amino acid sequence of the protein encoded by the gene is known, it is possible to work backward through the genetic code to the DNA sequence (Figure 13.12). Because the genetic code is degenerate (that is, several codons may specify the same amino acid; see

3

Place nitrocellulose filter in sealable plastic bag with solution of labeled DNA probe

4

Wash filter, prepare autoradiograph and compare with master plate

Radioactive probe will hybridize with

its complementary DNA

5

Darkening identifies colonies (plaques) containing the DNA desired

Autoradiograph film

● Cloning genes using oligonucleotide probes designed from a known amino acid sequence. A radioactively labeled set of DNA (degenerate) oligonucleotides representing all possible mRNA coding sequences is synthesized. (In this case, there are 25, or 32.) The complete mixture is used to probe the genomic library by colony hybridization (see Figure 13.11).

Biology of the Gene. Menlo Park, CA: Benjamin-Cummings.)

Nitrocellulose filter replica of bacterial colonies carrying different DNA fragments

2

Synthesize 32 possible DNA oligonucleotides and end label with radioactive 32P

3

Incubate nitrocellulose filter with probe solution in plastic bag

4

Hybridization of the correct oligonucleotide to the DNA

5

Detection by autoradiography

Autoradiograph film

Chapter 32), probes designed by this approach are usually degenerate oligonucleotides about 17 to 50 residues long (such oligonucleotides are so-called 17to 50-mers). The oligonucleotides are synthesized so that different bases are incorporated at sites where degeneracies occur in the codons. The final preparation thus consists of a mixture of equal-length oligonucleotides whose sequences vary to accommodate the degeneracies. Presumably, one oligonucleotide sequence in the mixture will hybridize with the target gene. These oligonucleotide probes are at least 17-mers because shorter degenerate oligonucleotides might hybridize with sequences unrelated to the target sequence.

A piece of DNA from the corresponding gene in a related organism can also be used as a probe in screening a library for a particular gene. Such probes are termed heterologous probes because they are not derived from the homologous (same) organism.

Problems arise if a complete eukaryotic gene is the cloning target; eukaryotic genes can be tens or even hundreds of kilobase pairs in size. Genes this size are fragmented in most cloning procedures. Thus, the DNA identified by the probe may represent a clone that carries only part of the desired gene. However, most cloning strategies are based on a partial digestion of the genomic DNA, a technique that generates an overlapping set of genomic fragments. This being so, DNA segments from the ends of the identified clone can now be used to probe the library for clones carrying DNA sequences that flanked the original isolate in the genome. Repeating this process ultimately yields the complete gene among a subset of overlapping clones.

cDNA Libraries

cDNAs are DNA molecules copied from mRNA templates. cDNA libraries are constructed by synthesizing cDNA from purified cellular mRNA. These libraries present an alternative strategy for gene isolation, especially eukaryotic genes. Because most eukaryotic mRNAs carry 3 -poly(A) tails, mRNA can be selectively isolated from preparations of total cellular RNA by oligo(dT)-cellulose chromatography (Figure 13.13). DNA copies of the purified mRNAs are synthesized by first annealing short oligo(dT) chains to the poly(A) tails. These oligo(dT) chains serve as primers for reverse transcriptase – driven synthesis of DNA (Figure 13.14). (Random oligonucleotides can also be used as primers, with the advantages being less dependency on poly(A) tracts and increased likelihood of creating clones representing the 5 -ends of mRNAs.) Reverse transcriptase is an enzyme that synthesizes a DNA strand, copying RNA as the template. DNA polymerase is then used to copy the DNA strand and form a dou- ble-stranded (duplex DNA) molecule. Linkers are then added to the DNA duplexes rendered from the mRNA templates, and the cDNA is cloned into a suitable vector. Once a cDNA derived from a particular gene has been identified, the cDNA becomes an effective probe for screening genomic libraries for isolation of the gene itself.

Because different cell types in eukaryotic organisms express selected subsets of genes, RNA preparations from cells or tissues in which genes of interest are selectively transcribed are enriched for the desired mRNAs. cDNA

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13.2 ● DNA Libraries 409

FIGURE 13.13 ● Isolation of eukaryotic mRNA via oligo(dT)-cellulose chromatography.

(a) In the presence of 0.5 M NaCl, the poly(A) tails of eukaryotic mRNA anneal with short oligo(dT) chains covalently attached to an insoluble chromatographic matrix such as cellulose. Other RNAs, such as rRNA (green), pass right through the chromatography column. (b) The column is washed with more

0.5 M NaCl to remove residual contaminants.

(c) Then the poly(A) mRNA is recovered by washing the column with water because the base pairs formed between the poly(A) tails of the mRNA and the oligo(dT) chains are unstable in solutions of low ionic strength.

EcoRI ended–cDNA duplexes for cloning

FIGURE 13.14 ● Reverse transcriptase – driven synthesis of cDNA from oligo(dT) primers annealed to the poly(A) tails of purified eukaryotic mRNA. (a) Oligo(dT) chains serve as primers for synthesis of a DNA copy of the mRNA by reverse transcriptase. Following completion of first-strand cDNA synthesis by reverse transcriptase, RNase H and DNA polymerase are added (b). RNase H specifically digests RNA strands in DNA RNA hybrid duplexes. DNA polymerase copies the first-strand cDNA, using as primers the residual RNA segments after RNase H has created nicks and gaps (c). DNA polymerase has a 5 n3 exonuclease activity that removes the residual RNA as it fills in with DNA. The nicks remaining in the second-strand DNA are sealed by DNA ligase (d), yielding duplex cDNA. EcoRI adapters with 5 -overhangs are then ligated onto the cDNA duplexes (e) using phage T4 DNA ligase to create EcoRIended cDNA for insertion into a cloning vector.

Any given DNA fragment is unique solely by virtue of its specific nucleotide sequence. The only practical way to find one particular DNA segment among a vast population of different DNA fragments (such as you might find in genomic DNA preparations) is to exploit its sequence specificity to identify it. In 1975, E. M. Southern invented a technique capable of doing just that.

Electrophoresis

Southern first fractionated a population of DNA fragments according to size by gel electrophoresis (see step 2 in figure). The electrophoretic mobility of a nucleic acid is inversely proportional to its molecular mass. Polyacrylamide gels are suitable for separation of nucleic acids of 25 to 2000 bp. Agarose gels are better if the DNA fragments range up to 10 times this size. Most preparations of genomic DNA show a broad spectrum of sizes, from less than 1 kbp to more than 20 kbp. Typically, no discrete-size fragments are evident following electrophoresis, just a “smear” of DNA throughout the gel.

Blotting

Once the fragments have been separated by electrophoresis (step 3), the gel is soaked in a solution of NaOH. Alkali denatures duplex DNA, converting it to single-stranded DNA. After the pH of the gel is adjusted to neutrality with buffer, a sheet of nitrocellulose soaked in a concentrated salt solution is then placed over the gel (c), and salt solution is drawn through the gel in a direction perpendicular to the direction of electrophoresis (step 4). The salt solution is pulled through the gel in one of three ways: capillary action (blotting), suction (vacuum blotting), or electrophoresis (electroblotting). The movement of salt solution through the gel carries the DNA to the nitrocellulose sheet. Nitrocellulose binds single-stranded DNA molecules very tightly, effectively immobilizing them in place on the sheet.* Note that the distribution pattern of the electrophoretically separated DNA

The Southern blotting technique involves the transfer of electrophoretically separated DNA fragments to a nitrocellulose sheet and subsequent detection of specific DNA sequences. A preparation of DNA fragments [typically a restriction digest, (1)] is separated according to size by gel electrophoresis (2). The separation pattern can be visualized by soaking the gel in ethidium bromide to stain the DNA and then illuminating the gel with UV light (3). Ethidium bromide molecules intercalated between the hydrophobic bases of DNA are fluorescent under UV light. The gel is soaked in strong alkali to denature the DNA and then neu-

is maintained when the single-stranded DNA molecules bind to the nitrocellulose sheet (step 5 in figure). Next, the nitrocellulose is dried by baking in a vacuum oven;† baking tightly fixes the single-stranded DNAs to the nitrocellulose. Next, in the prehybridization step, the nitrocellulose sheet is incubated with a solution containing protein (serum albumin, for example) and/or a detergent such as sodium dodecyl sulfate. The protein and detergent molecules saturate any remaining binding sites for DNA on the nitrocellulose. Thus, no more DNA can bind nonspecifically to the nitrocellulose sheet.

Hybridization

To detect a particular DNA within the electrophoretic smear of countless DNA fragments, the prehybridized nitrocellulose sheet is incubated in a sealed plastic bag with a solution of specific probe molecules (step 6 in figure). A probe is usually a single-stranded DNA of defined sequence that is distinctively labeled, either with a radioactive isotope (such as 32P) or some other easily detectable tag. The nucleotide sequence of the probe is designed to be complementary to the sought-for or target DNA fragment. The singlestranded probe DNA anneals with the single-stranded target DNA bound to the nitrocellulose through specific base pairing to form a DNA duplex. This annealing, or hybridization as it is usually called, labels the target DNA, revealing its position on the nitrocellulose. For example, if the probe is 32P-labeled, its location can be detected by autoradiographic exposure of a piece of X-ray film laid over the nitrocellulose sheet (step 7 in figure).

Southern’s procedure has been extended to the identification of specific RNA and protein molecules. In a play on Southern’s name, the identification of particular RNAs following separation by gel electrophoresis, blotting, and probe hybridization is called Northern blotting. The analogous technique for identifying protein molecules is termed Western blotting. In Western blotting, the probe of choice is usually an antibody specific for the target protein.