Molecular Biotechnology: Principles and Applications - Glick, B., Pasternak, J. 2002

Fundamentals of Molecular Biotechnology
Chemical synthesis, nucleotide sequence determination, and DNA amplification
Polymerase chain reaction

The Polymerase Chain Reaction (PCR) is an efficient method for generating A large number of copies of specific nucleotide sequences in vitro. Their Amplification—sometimes by a factor of millions—is carried out through a three-stage cyclic process. PCR requires: 1) two synthetic oligonucleotide primers (approximately 20 NUCLEOTIDES in length) complementary to DNA regions from opposite strands that flank the target sequence, with their 3'-hydroxyl ends oriented toward each other after annealing to the DNA; 2) a target DNA ranging from 100 to ~35,000 bp in length; 3) a thermostable DNA polymerase that retains its activity at 95° and above; and 4) four deoxynucleotides.

A typical PCR amplification consists of the repeated execution of the following three reactions.

1. Denaturation. The first step of PCR involves thermal denaturation of the DNA sample by holding it at 95 °C for at least 1 min. In addition to the DNA, the reaction mixture contains an excess of two primers, the thermostable Taq DNA polymerase isolated from the bacterium Thermus aquaticus, and four deoxynucleotides.

2. Renaturation. The Temperature of the mixture is slowly lowered to ~55 °C, allowing the primers to pair with their complementary DNA sequences.

3. Synthesis. The temperature is raised to ~75 °C, which is the optimum temperature for Taq DNA polymerase. The Synthesis of the complementary DNA strand begins, initiated by the 3'-hydroxyl group of the primer (Fig. 5.18).

All reactions are carried out in tubes immersed in a thermostat. The temperature regime is changed and maintained automatically. Each cycle typically lasts 3–5 min. To understand exactly how a specific DNA segment is amplified during PCR, one must clearly visualize the positions of all primers and their complementary sequences within the amplified strands in each round. In the first round, each of the newly synthesized strands is much longer than the distance from the 3'-hydroxyl group of "its" primer to the terminal nucleotide of the sequence complementary to the second primer. Such strands are called "long templates," and it is upon them that subsequent synthesis will take place (Fig. 5.18).

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Fig. 5.18. First round of PCR. The target DNA is flanked by sequences 1'-2 in one strand and sequences 1-2' in the other. Primers (P1 and P2), Taq DNA polymerase, and four deoxynucleoside triphosphates (dNTPs) are added to the DNA sample. The mixture is heated to 95 °C, incubated for 1 min, and slowly cooled to 55 °C. At this temperature, the primers, added in excess, pair with the separated strands. The temperature is then raised to 75 °C. Under these conditions, the synthesis of both DNA strands takes place, initiating from the 3'-hydroxyl ends of the primers. Each synthesized strand is much longer than the distance from the 3'-hydroxyl group of "its" primer to the terminal nucleotide of the sequence complementary to the second primer. These strands serve as templates In the second round of PCR.

In the second round, the double-stranded DNA consisting of the original and newly synthesized ("long template") strands is denatured again and then annealed with primers. During the synthesis phase of this round, "long templates" are synthesized anew, along with a certain number of strands possessing a primer at one end and a sequence complementary to the second primer at the other ("short templates") (Fig. 5.19). During the third round, all previously formed heteroduplexes are simultaneously subjected to denaturation and primer annealing, followed by Replication (Fig. 5.20). In subsequent rounds, the number of "short templates" increases progressively, and by the 30th round, their number exceeds that of the original strands or "long templates" by a factor of 10⁶ (Fig. 5.21).

The PCR method has become widely adopted. We will discuss its diverse Applications in subsequent chapters, mentioning only a few here. One of the most important is the identification of pathogenic microorganisms that cause diseases in humans, animals, and plants. With the advent of PCR, the Isolation and Purification of target DNA are no longer necessary; very small amounts of crude material can be used for analysis. To synthesize primers specific exclusively to the target DNA, The nucleotide sequence of the putative pathogen's DNA must be known. In this case, PCR will amplify only a DNA fragment whose length equals the combined length of the two primers plus the DNA fragment between them.

Because PCR is a highly sensitive method, even a minuscule amount of DNA accidentally transferred from one reaction mixture to another in the test sample can yield false-positive results. This necessitates rigorous quality control of all solutions and glassware used for PCR.

Fig. 5.19. Second round of PCR. The starting material in this case is the mixture of DNA molecules generated in the first round (Fig. 5.18). During annealing, the primers hybridize to complementary regions on both the original strands and the "long templates" synthesized in the first round. As a result of in vitro enzymatic synthesis, "long templates" are synthesized on the original strands, and "short templates" are synthesized on the "long templates." The latter begin with one primer and end with a sequence complementary to the second primer.

Fig. 5.20. Third round of PCR. During annealing, the primers hybridize to complementary Regions of the original strands as well as the "long" and "short" templates. During in vitro enzymatic synthesis, "long" templates are synthesized on the original strands, whereas only "short templates" are synthesized on the "long" and "short" templates.

Fig. 5.21. Thirtieth round of PCR. At this stage, the reaction mixture consists almost exclusively of "short templates."

The PCR method is also used to detect spontaneous Mutations, introduce specific mutations in vitro, assemble full-length genes from synthetic oligonucleotides, and perform DNA Sequencing. In many cases, it is necessary to clone the PCR product. However, direct cloning via blunt-end ligation is hindered because Taq polymerase adds an extra adenine nucleotide to the 3'-end of the synthesized strand, which reduces ligation efficiency. Nevertheless, if the cloning vector is treated with a restriction endonuclease to generate fresh blunt ends and then incubated with Taq polymerase in the presence of dTTP, a single thymidine nucleotide is added to both 3'-ends of the fragments. A single-nucleotide complementarity between the ends of the vector and the PCR product is sufficient for the molecules to anneal and subsequently undergo ligation.

Generating cDNA Corresponding to the Ends of mRNA Molecules Using PCR

PCR can be used to obtain complementary DNAs (cDNAs) corresponding to the 3'- or 5'-terminal regions of specific messenger RNAs (mRNAs). This method is abbreviated as RACE, which stands for rapid amplification of cDNA ends (rapid

Fig. 5.22. PCR amplification of cDNA complementary to the 3'-terminal portion of mRNA. The first cDNA strand is generated by reverse METABOLISM/31.html">Transcription of mRNA using oligo(dT) as a primer. The second strand is synthesized using the first strand as a template with Taq polymerase and a Gene-specific primer (GSP1). Primers GSP2 and P are used in subsequent rounds of PCR.

cDNA end amplification). The designations 3' RACE and 5' RACE refer to the amplification of cDNAs corresponding to the respective ends of the mRNA. In both cases, performing PCR amplification requires knowing the nucleotide sequence of the coding region of the target mRNA in order to synthesize a gene-specific primer (GSP). For 3' RACE, the primer for synthesizing the first cDNA strand is an oligo(dT) with an attached second primer (P) (Fig. 5.22). The oligo(dT) anneals to the poly(A) tail of the mRNA, and Reverse Transcriptase synthesizes a strand complementary to the mRNA. The second cDNA strand is synthesized on the first strand using Taq polymerase and a GSP complementary to the coding region of the given mRNA. After several rounds of PCR using the aforementioned primers, a second pair of primers is added that bind adjacent to the first two. Such closely spaced primers are called nested primers. The second pair of primers is required because the full-length target molecule cannot be amplified without them. The final PCR product is a cDNA corresponding to the 3'-end of the desired mRNA.

Fig. 5.23. PCR amplification of cDNA complementary to the 5'-terminal part of the mRNA first strand, carried out by reverse transcriptase, is initiated by the GSP primer. Next, a poly(A) tail is added to this strand using terminal deoxynucleotidyl transferase. Oligo(dT) is used as a primer for second-strand synthesis. Several rounds of amplification are performed using the specified primers, a second set of primers (GSP2 and P) is added, and a cDNA corresponding to the 5'-end of the mRNA is obtained.

Fig. 5.24. Gene synthesis by PCR. Overlapping oligonucleotides (A and B) are annealed and the resulting duplex with recessed 3'-hydroxyl ends is extended. The double-stranded molecules are denatured, a second pair of oligonucleotides (C and D) overlapping with the products of the first PCR round is added, and annealing is carried out. A second round of PCR is performed, the next pair of oligonucleotides (E and F) is added, a third round of PCR is carried out, and so on. As a result, a double-stranded DNA molecule identical to the target gene is formed. Identical letters with or without a prime (A' and A, B and B', etc.) designate complementary DNA regions. The nucleotide sequence of each oligonucleotide corresponds to that of specific DNA segments.

In the case of 5'RACE, the GSP serves as the primer for first-strand cDNA synthesis (Fig. 5.23). The newly synthesized strand is treated with terminal deoxynucleotidyl transferase in the presence of dATP. This enzyme randomly adds deoxyribonucleotides to the 3'-end of the strand. Since only dATP is present in the reaction mixture in this case, a stretch of adenine residues—a poly(A) tail—is formed at this end. The P-oligo(dT) primer anneals to this tail and initiates second-strand synthesis. A limited number of PCR rounds are performed with the indicated primers, followed by The addition of nested primers to amplify the cDNA corresponding to the 5'-end of the mRNA.

The RACE method is widely used for several reasons. It is usually very difficult to detect cDNA corresponding to an mRNA present in low Abundance in a given tissue. The RACE method allows the rapid generation of cDNAs corresponding to the terminal regions of such mRNA and, if necessary, their use as probes for screening cDNA and Genomic Libraries. Furthermore, since incomplete 3'-cDNA fragments significantly outnumber full-length ones, 5'RACE can fill in the missing 5'-terminal segments.

Gene Synthesis by PCR

Obtaining genes via PCR is a much faster and more cost-effective method than the one based on annealing overlapping oligonucleotides, filling in gaps with DNA polymerase, and sealing the nicks with DNA ligase. In one approach, gene construction begins with the annealing of two overlapping oligonucleotides (A and B) corresponding to the central part of the gene (Fig. 5.24). Annealing yields a duplex with recessed 3'-hydroxyl groups that serve as initiation sites for the synthesis of complementary strands during PCR. Next, two additional oligonucleotides, C and D, are added to the reaction mixture. The 3'-end of oligonucleotide C is identical to the 5'-end of oligonucleotide A, and the oligonucleotide itself corresponds to the region of the gene being constructed that immediately flanks the left side of its central part. Similarly, the 3'-end of oligonucleotide D is identical to the 5'-end of oligonucleotide B and corresponds to the region of the gene flanking the right side of its central part. Following mixture denaturation and annealing, duplexes with extended protruding single-stranded segments are formed, which are then extended from their 3'-ends. During subsequent PCR rounds, a double-stranded product is formed, consisting of the aforementioned segments arranged in the order CABD. DNA molecules with recessed 5'-ends are not extended.

At the next stage, two more oligonucleotides, E and F, are added to the reaction mixture. The 3'-end of oligonucleotide E is identical to the 5'-end of oligonucleotide C, and it corresponds to the region of the reconstructed gene flanking segment C on the left. Oligonucleotide F exhibits analogous properties relative to oligonucleotide D. Following denaturation and renaturation of the mixture, the resulting duplexes with protruding single-stranded regions are extended from their 3'-hydroxyl ends. During subsequent PCR rounds, the double-stranded product ECABDF is formed.

Subsequent pairs of oligonucleotides—one extending the gene on the left, the other on the right—are sequentially added to the mixture until the entire gene is synthesized. The length of these oligonucleotides is typically 50 bases. Each PCR "block" consists of twenty 4-minute rounds. Synthesizing a 1000 bp gene requires 10 such blocks, allowing the gene to be obtained within a single day. Moreover, as with other gene synthesis Methods, the final pair of nucleotides (i.e., the 5'- and 3'-ends) can be endowed with additional sequences flanking the coding region to facilitate subsequent insertion of the gene into a vector.