Molecular Biotechnology. Bernard R. Glick

primer and the template molecule is sufficiently stable for primer extension by DNA polymerase. At the end of the first cycle of PCR, the noncomplementary regions of the primer remain single stranded in the otherwise double-stranded DNA product. However, after the extension step of the second cycle, the newly synthesized complementary strand extends to the 5′ end of the primer sequence on the template strand and therefore the PCR product contains a double-stranded restriction enzyme recognition site at one end (Fig. 2.21). Subsequent cycles yield DNA products with double-stranded restriction enzyme sites at both ends that can be cleaved to generate sticky ends for insertion into a vector. Alternatively, PCR products can be cloned using the single deoxyadenosine monophosphate (dAMP) that is added to the 3′ ends by Taq DNA polymerase, which lacks the proofreading activity of many DNA polymerases to correct mispaired bases. A variety of linearized vectors have been constructed that possess a single complementary 3′ deoxythymidine monophosphate (dTMP) overhang to facilitate cloning without using restriction enzymes (Fig. 2.22).

      Figure 2.21 Addition of restriction enzyme recognition sites to PCR-amplified target DNA to facilitate cloning. Each of the two oligonucleotide primers (P1 and P2) has a sequence of approximately 20 nucleotides in the 3′ end that is complementary to a region flanking the target DNA (shown in black). The sequence at the 5′ end of each primer consists of a restriction endonuclease recognition site (shown in green) that does not base-pair with the template DNA during the annealing steps of the first and second PCR cycles. However, during the second cycle, the long DNA strands produced in the first cycle serve as templates for synthesis of short DNA strands (indicated by a terminal arrow) that include the restriction endonuclease recognition sequences at both ends. DNA synthesis during the third and subsequent PCR cycles produces double-stranded DNA molecules that carry the target DNA sequence flanked by restriction endonuclease recognition sequences. These linear PCR products can be cleaved with the restriction endonucleases to produce sticky ends for ligation with a vector. Note that not all of the DNA produced during each PCR cycle is shown.

      Figure 2.22 Cloning of PCR products without using restriction endonucleases. Taq DNA polymerase adds a single dAMP (A) to the ends of PCR-amplified DNA molecules. These extensions can base-pair with complementary single dTMP (T) overhangs on a specially constructed linearized cloning vector. Ligation with T4 DNA ligase results in insertion of the PCR product into the vector.

Quantitative PCR

      PCR protocols have been developed to quantify the number of target DNA molecules, or RNA molecules after conversion to cDNA, present in a sample. Quantitative PCR is based on the principle that under optimal conditions, the number of DNA molecules doubles after each cycle. Typically, the amount of DNA present after each PCR cycle is measured in real time as the amount of fluorescence emitted by a fluorescent dye bound to the double-stranded DNA product. Thus, the fluorescence intensity increases in proportion to the concentration of double-stranded DNA (Fig. 2.23).

      Figure 2.23 The fluorescent dye SYBR green does not bind to single-stranded DNA (A), binds to double-stranded DNA as it is synthesized (B), and is bound to the double-stranded amplified DNA (C). Only the dye-bound DNA fluoresces.

      A real-time PCR occurs in four phases (Fig. 2.24A). In the first, or linear, phase (generally about 10 to 15 cycles), fluorescence emission at each cycle has not yet risen above the background level and therefore cannot be quantified accurately. In the second, or early exponential, phase, a sufficient amount of double-stranded DNA has been produced to increase the amount of fluorescence above a threshold level that is significantly higher than the background. The cycle at which this occurs is known as the threshold cycle (CT). The CT value is inversely correlated with the amount of target DNA in the original sample. During the third, exponential phase, the amount of fluorescence continues to double as the DNA products of the reaction double in each cycle under ideal conditions. However, in the final, plateau phase, the reaction components become limited and measurements of the fluorescence intensity are no longer useful.

      Figure 2.24 (A) Plot of normalized fluorescence (ΔRn) versus cycle number in a real-time PCR experiment. Four phases of PCR are shown: (1) a linear phase, where fluorescence emission is not yet above background level; (2) an early exponential phase, where the fluorescence intensity becomes significantly higher than the background (the cycle at which this occurs is generally known as CT); (3) an exponential phase, where the amount of product doubles in each cycle; and (4) a plateau phase, where reaction components are limited and amplification slows down. (B) Plot of CT versus the starting amount of a target nucleotide sequence. Fluorescence detection is linear over several orders of magnitude.

      To quantify the amount of target DNA in a test sample, a standard curve is first generated by serially diluting a control sample with a known number of copies of the target DNA, and assuming all dilutions are amplified with equal efficiency, the CT values for each dilution are plotted against the known starting amount of DNA (Fig. 2.24B). The number of copies of a target DNA in a test sample can then be determined by obtaining the CT value for the test sample and extrapolating the starting amount from the standard curve. Since the amount of DNA doubles with each cycle during the exponential phase, a sample that has four times the number of starting copies of the target sequence compared to another sample would require two fewer cycles of amplification to generate the same number of product strands. Often, a melt curve is generated to assess the specificity of the products, which denature at a characteristic temperature that is determined by their nucleotide sequence.

      Among its many applications, quantitative real-time PCR has been used to monitor microorganisms that cause a range of infectious diseases. For example, it has been used to quantify Salmonella enterica contamination in food samples. In this case, food samples (chicken and mung beans were tested) were rinsed with water or a saline solution, and the liquid was filtered to collect the bacterial cells. The bacterial cells were removed from the filter membrane, lysed, and subjected to real-time PCR. In this case, the entire procedure took only approximately 3 hours and was able to detect and quantify as few as 700 S. enterica cells per 100 ml of liquid.

Chemical Synthesis of DNA

      The ability to chemically synthesize DNA with a specific sequence of nucleotides easily, inexpensively, and rapidly is essential for many of the methodologies of molecular biotechnology. Chemically synthesized, single-stranded DNA oligonucleotides (10−100 nucleotides) are used for amplifying specific DNA sequences by PCR, introducing mutations into cloned genes, sequencing DNA, and synthesizing whole genes and chromosomes (see box on next page).

box Synthetic Genomes

      Chemically synthesized oligonucleotides have been assembled not only into genes but also into whole genomes. The first genome to be produced synthetically was the cDNA encoding the small (7,500 bp) single-stranded RNA genome of poliovirus (Cello et al., Science. 297:1016−18, 2002). The poliovirus genome sequence was known and facilitated the design of 70 nucleotide-long, single-stranded oligonucleotides. Overlapping complementarity at the termini of neighboring oligonucleotides enabled their assembly into 400 to 600 bp fragments. The fragments were ligated into three larger segments that were subsequently digested with a restriction endonuclease and cloned in the correct order and orientation into a plasmid. Expression of the full-length cDNA