Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Ogurtsov 2011

Identification of DNA Clones
Polymerase Chain Reaction

11.3.1. Amplification of DNA fragments. If The nucleotide sequences at the ends of a given DNA region are known, this entire region of the DNA molecule can be replicated many times using the Polymerase chain reaction (PCR). Such multiple Replication is called amplification (from the English word "amplify").

PCR utilizes the property of double-stranded DNA to reversibly denature (melt) into two separate strands upon Temperature change (see Figure 4). Much like in membrane Hybridization, the presence of non-complementary DNA strands in the solution does not prevent the base-pairing of complementary DNA strands or individual complementary Regions of the strands.

The second essential requirement for PCR is the availability of pre-synthesized primers—oligonucleotides 18–20 NUCLEOTIDES in length with a predefined sequence. Typically, such oligonucleotides are synthesized using automated synthesizers based on the standard scheme shown in Figure 94.

The General scheme of PCR is illustrated in Figure 103.

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Figure 103 — Scheme of the polymerase chain reaction

PCR begins with the thermal Denaturation of DNA into two single strands. Next, two pre-synthesized oligonucleotides (primers) complementary to the 3' ends of the single strands of the targeted DNA region to be amplified are added to the solution. Here, it is crucial that the concentration of primers in the solution significantly exceeds the concentration of the single-stranded DNA templates to be amplified. In Figure 103, primers are depicted as black and white rectangles.

The solution is then cooled to 50–60°C. This allows the primers (whose concentration is much higher than that of the DNA) to hybridize (anneal) with the complementary regions of the DNA strands near the 3' ends. Due to their low concentration, the single DNA strands themselves do not yet renature at this temperature.

These hybridized oligonucleotides then function as primers for the synthesis of complementary DNA strands, using the original single-stranded DNA as templates.

For this process, the solution must contain an excess of:

1) deoxynucleotides,

2) thermostable DNA polymerases.

An example of such a DNA polymerase is the Taq polymerase from the thermophilic bacterium Thermus aquaticus. Because Thermus aquaticus lives in hot springs, Taq polymerase retains its globular Structure and activity even when heated up to 95°C and can initiate the polymerization reaction at 72°C, starting from the synthetic oligonucleotide primer hybridized to the DNA.

Once the Synthesis of the complementary strands is complete, the mixture is heated to 95°C to melt (denature) the newly synthesized DNA duplexes.

Afterwards, since an excess of oligonucleotide primers remains in the solution, cooling the mixture to 50–60°C leads to primer rehybridization with the DNA strands, and a new PCR cycle begins.

Repeating the cycles of melting (upon heating) and synthesis (after cooling), in each of which the number of synthesized DNA segments doubles, leads to a dramatic increase in the concentration (amplification) of the target DNA region flanked by the primers. After 20 cycles of PCR amplification, the number of initial molecules increases a million-fold (2²⁰ = 1,048,576), while other regions of the original DNA molecule remain unamplified.

11.3.2. ISOLATION OF A specific genomic DNA segment. For organisms whose genome has been fully sequenced, the simplest way to clone a specific genomic DNA fragment is typically a Procedure that starts with PCR using a mixture containing the entire genomic DNA. In this case, two oligonucleotide primers are first synthesized that:

1) are capable of hybridizing to the DNA on both sides of the region of interest,

2) contain the nucleotide sequences recognized by specific restriction Enzymes (Figure 104).

Figure 104 — PCR of a DNA fragment for cloning into a plasmid vector

As early as the third PCR cycle, only the DNA fragments located between the primers will be amplified. Following the amplification of the required quantity of these DNA fragments (~20 PCR cycles), the mixture is treated with restriction enzymes that generate sticky ends on the DNA fragments. This allows such DNA fragments to be efficiently inserted into the polylinkers of Plasmid Vectors cut with the same restriction enzymes (Figure 104). The recombinant Plasmids generated by this method can then be cloned in E. coli Cells.

A key feature of this method is that it eliminates the need to clone A large number of diverse (and generally randomly generated) restriction fragments of the initial genomic DNA, followed by screening to identify and isolate the exact fragment under investigation.

In effect, the PCR technique inverts the traditional workflow—the Selection of the specific fragment is performed prior to cloning, rather than after.

The PCR technique is used to isolate specific Gene sequences for subsequent manipulation. It can also be employed to locate and isolate gene sequences from a mutant Organism to determine how they differ from the original wild-type genome.

11.3.3. Preparation of DNA Probes by PCR. Oligonucleotide probes for screening via hybridization can be generated using PCR amplification. In this approach, the only prerequisite is the Chemical synthesis of two relatively short primers corresponding to the two ends of the target nucleotide sequences specific to each probe.

Either genomic DNA or processed mRNA can serve as the template for PCR amplification. In the latter case, a comprehensive set of cDNAs is synthesized from the entire mRNA pool using Reverse Transcriptase, or alternatively, a pooled mixture of cDNAs is generated from all clones of a λ-cDNA library.

To produce radiolabeled probes via PCR, dNTPs labeled with radioactive phosphorus ³²P are incorporated during the final few amplification cycles. Because PCR-generated probes are relatively long, they incorporate a large number of ³²P radioactive atoms. As a result, these PCR probes yield a stronger and much more specific signal compared to chemically synthesized probes.