sgRNA contains a 20-nucleotide sequence (hatched region) that is complementary to the target site in the host genome. Promoters (arrows) for the sgRNA and Cas9 genes, and codon usage for Cas9, must be suitable for expression in host cells. An origin of replication (ori) and a selectable marker (e.g., bla encoding β-lactamase, which confers resistance to the antibiotic ampicillin) are included for initial vector construction in E. coli. The vector and donor DNA are introduced into a recipient cell. Following expression, the sgRNA guides the Cas9 endonuclease to the target sequence in the recipient cell chromosome and the endonuclease makes a double-stranded break in the target DNA. (B) The donor DNA sequence (green) is flanked by regions that are homologous to the target site (grey) for insertion by homologous recombination. Therefore, activation of recombinases that mediate DNA repair results in recombination between homologous sequences on the vector and in the recipient chromosome, and thereby, insertion of the donor DNA into the genome at the target site.
Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a simple, efficient procedure for synthesizing large quantities of a specific DNA sequence in vitro (see Milestone box on page 36). The reaction exploits the mechanism used by living cells to accurately replicate a DNA template. PCR can be used to produce millions of copies from a single template molecule and to detect a specific sequence in a complex mixture of DNA.
milestone Specific Enzymatic Amplification of DNA In Vitro: the Polymerase Chain Reaction
PCR, which is the invention of Kary Mullis (U.S. patent 4,683,202), has had a tremendous impact on many research areas, including molecular biotechnology. The power of the method is in its simplicity, sensitivity, and specificity. It utilizes a mechanism similar to that used by our cells to accurately replicate a DNA template, it can detect and produce millions of copies from a single template molecule in a few hours, and, under appropriate conditions, it can amplify a specific sequence in a complex mixture of DNA molecules even when other similar sequences are present.
PCR was a unique idea that did not replace any existing technology. In the early 1980s, Kary Mullis was trying to solve the problem of using synthetic oligonucleotides to detect single nucleotide mutations in sequences that were present in low concentration. He needed a method to increase the concentration of the target sequence. He reasoned that if he mixed heat denatured DNA with two oligonucleotides that bound to opposite strands of the DNA at an arbitrary distance from each other and added some DNA polymerase and deoxynucleoside triphosphates, the polymerase would add the deoxynucleoside triphosphates to the hybridized oligonucleotides. The reaction did not yield the expected products. Mullis then heated the reaction products to separate the extended oligonucleotides from the template DNA and then repeated the process with fresh polymerase, hypothesizing that after each cycle the number of molecules carrying the specific sequence between the primers would double. Despite the skepticism of his colleague, Mullis proved that his reasoning was correct, albeit the hard way. By manually cycling the reaction through temperatures required to denature the DNA and anneal and extend the oligonucleotides, each time adding a fresh aliquot of a DNA polymerase isolated from E. coli, he was able to synthesize unprecedented amounts of target DNA (Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51:263–273, 1986). Thermostable DNA polymerases that obviate the need to add fresh polymerase after each denaturation step and automated cycling have since made PCR a routine and indispensable laboratory procedure.
The capability of generating large amounts of DNA by amplification from segments of cloned or genomic DNA has facilitated the cloning of DNA versions of rare mRNA molecules, screening gene libraries, diagnostic testing for gene mutations, sequencing of genomes, and a myriad of other applications. In fact, the first study using PCR described a diagnostic test for sickle-cell anemia (Saiki et al., Science. 230:1350–1354, 1985). Mullis received the Nobel Prize in Chemistry for his work on PCR in 1993.
Amplification of DNA Using PCR
The essential components for PCR amplification are (i) a template sequence in a DNA sample that is targeted for amplification and is from 100 to 3,000 bp in length (larger regions can also be amplified, but with reduced efficiency); (ii) two synthetic oligonucleotide primers (∼20 nucleotides each) that are complementary to regions on opposite strands that flank the target DNA sequence and that, after annealing to the sample DNA, have their 3′ hydroxyl ends oriented toward each other; (iii) a thermostable DNA polymerase that remains active after repeated heating to 95°C or higher and copies the DNA template with high fidelity; (iv) the four deoxyribonucleotides; and (v) a reaction buffer that provides optimal pH and osmotic conditions, and cofactors (e.g., magnesium) required for DNA polymerase activity.
Replication of a specific DNA sequence by PCR requires three successive steps as outlined below. Amplification is achieved by repeating the three-step cycle 25 to 40 times. All steps in a PCR cycle are carried out in an automated block heater that is programmed to change temperatures after a specified period of time.
1 Denaturation. The first step in a PCR is the thermal denaturation of the double-stranded DNA template to separate the strands. This is achieved by raising the temperature of a reaction mixture to 95°C. The reaction mixture is comprised of the sample DNA that contains the target DNA to be amplified, a vast molar excess of the two oligonucleotide primers, a thermostable DNA polymerase (e.g., Taq DNA polymerase, isolated from the bacterium Thermus aquaticus), four deoxyribonucleotides, and the reaction buffer.
2 Annealing. For the second step, the temperature of the mixture is slowly cooled. During this step, the primers base-pair, or anneal, with their complementary sequences in the DNA template. The temperature at which this step of the reaction is performed is determined by the nucleotide sequence of the primer that forms hydrogen bonds with complementary nucleotides in the target DNA. Typical annealing temperatures are in the range of 45 to 68°C, although optimization is often required to achieve the desired outcome, that is, a product consisting of fragments of target DNA sequence only.
3 Extension. In the third step, the temperature is raised to ∼70°C, which is optimal for the catalytic activity of Taq DNA polymerase. DNA synthesis is initiated at the 3′ hydroxyl end of each annealed primer, and nucleotides are added to extend the complementary strand using the sample DNA as a template.
To understand how the PCR protocol succeeds in amplifying a discrete segment of DNA, it is important to keep in mind the location of each primer annealing site and its complementary sequence within the strands that are synthesized during each cycle. During the extension phase of the first cycle, the newly synthesized DNA from each primer is extended beyond the endpoint of the sequence that is complementary to the second primer. These new strands form “long templates” that are used in the second cycle (Fig. 2.20).
Figure 2.20 PCR. During a PCR cycle, the template DNA is denatured by heating and then slowly cooled to enable two primers (P1 and P2) to anneal to complementary (black) bases flanking the target DNA. The temperature is raised to about 70°C, and in the presence of the four deoxyribonucleotides, Taq DNA polymerase catalyzes the synthesis of a DNA strand extending from the 3′ hydroxyl end of each primer. In the first PCR cycle, DNA synthesis continues past the region of the template DNA strand that is complementary to the other primer sequence. The products of this reaction are two long strands of DNA that serve as templates for DNA synthesis during the second PCR cycle. In the second cycle, the primers hybridize to complementary regions in both the original strands and the long template strands, and DNA synthesis produces more long DNA strands from the original strands and short strands from the long template strands. A short template strand has a primer sequence at one end and the sequence complementary to the other primer at its other end. During the third PCR cycle, the primers hybridize to complementary regions of original, long template, and short template strands, and DNA synthesis produces
