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 from a suitable promoter in HeLa cells resulted in production of viral RNA and proteins that were assembled into infectious poliovirus particles.
Construction of a much larger synthetic bacterial genome presented a greater challenge (Gibson et al., Science. 319:1215–20, 2008). The genome of the bacterium Mycoplasma genitalium was chosen because it has the smallest genome (a single chromosome of 580,076 bp) currently known for a free-living bacterium. The chromosome was initially produced in 101 segments of about 5 to 7 kb that were each assembled from synthetic oligonucleotides. The sequence of each segment overlapped its neighbor by approximately 80 bp and therefore, following brief treatment with an exonuclease to generate sticky ends, four neighboring segments were joined in vitro by complementary base-pairing at their termini and enzymatic repair of gaps. These 24-kb fragments were cloned into a bacterial artificial chromosome, which can carry large DNA inserts, and propagated in E. coli. This in vitro recombination method was repeated to assemble the fragments into successively larger pieces. Large segments carrying half- and full-genome sequences could not be cloned in E. coli and therefore quarter- and then half-genome fragments were finally assembled into a 582,970 bp sequence in the yeast Saccharomyces cerevisiae by in vivo recombination between overlapping homologous sequences.
These results demonstrated that a small bacterial genome can be constructed entirely from synthetic oligonucleotides. The next step was to show that a genome produced “from scratch” can direct the survival and growth of a living bacterium. The 1,077,947 bp genome of Mycoplasma mycoides was synthesized in a manner similar to that used to construct the M. genitalium genome (Gibson et al., Science 329:52–56, 2010); M. mycoides was chosen because it grows at a faster rate than the extremely slow growing M. genitalium. Briefly, overlapping synthetic oligonucleotides were assembled into 1,080 bp fragments. These fragments were recombined into 10-kb and then into 100-kb segments, and finally into a full-length genome by in vivo homologous recombination in yeast. The intact M. mycoides genome was extracted from yeast and transplanted into a related species Mycoplasma capricolum, replacing the recipient cell’s chromosome. Remarkably, the synthetic genome was self-replicating and controlled the functions of a living bacterium. The bacterium was able to grow logarithmically and exhibited cellular and colony morphologies similar to M. mycoides. “Watermark” sequences were inserted in four regions to enable differentiation of the synthetic and natural genomes.
A major motivation for producing a synthetic bacterium is to understand the minimal genetic requirements for life, that is, to identify the minimum set of essential genes that can support survival and reproduction of a cell. Because it takes less energy and fewer resources to maintain and propagate a small genome, more resources can be directed to the synthesis of high yields of useful products from cloned genes.
Synthesis of Oligonucleotides
Currently, the phosphoramidite method is the procedure of choice for chemical DNA synthesis. Solid-phase synthesis, in which the growing DNA strand is attached to a solid support, is used so that all the reactions can be conducted in one reaction vessel, the reagents from one reaction step can be readily washed away before the reagents for the next step are added, and the reagents can be used in excess in an attempt to drive the reactions to completion.
The chemical synthesis of DNA is a multistep process (Fig. 2.25). It does not follow the biological direction of DNA synthesis; rather, during the chemical process, each incoming nucleotide is coupled to the 5′ hydroxyl terminus of the growing chain. Before their introduction into the reaction column, the amino groups of the nucleotides’ nitrogenous bases adenine, guanine, and cytosine are derivatized by the addition of benzoyl, isobutyryl, and benzoyl groups, respectively, to prevent undesirable side reactions during polymerization. Thymine is not treated because it lacks an amino group. The initial nucleoside (base and sugar only), which will be the 3′-terminal nucleotide of the final synthesized strand, is attached to a spacer molecule by its 3′ hydroxyl terminus and the spacer molecule is covalently attached to an inert support, which is often a controlled pore glass (CPG) bead (a glass bead with uniformly sized pores) (Fig. 2.26). A dimethoxytrityl (DMT) group is attached to the 5′ end of the first nucleoside to prevent the 5′ hydroxyl group from reacting nonspecifically before the addition of the second nucleotide. Each nucleotide that is added to the growing chain has a 5′ DMT protective group and also a diisopropylamine group attached to a 3′ phosphite group that is protected by a β-cyanoethyl (CH2CH2CN) group (Fig. 2.27). This molecular assembly is called a phosphoramidite.
Figure 2.25 Flowchart for the chemical synthesis of DNA oligonucleotides. After n coupling reactions (cycles), a single-stranded
