From Genes to Genomes:- The Polymerase Chain Reaction Amplifies Specific DNA Sequences

The Human Genome Project, along with the many as sociated efforts to sequence the genomes of organisms of every type, is providing unprecedented access to gene sequence information. This in turn is simplifying the process of cloning individual genes for more detailed biochemical analysis. If we know the sequence of at least the flanking parts of a DNA segment to be cloned, we can hugely amplify the number of copies of that DNA segment, using the polymerase chain reaction (PCR),a process conceived by Kary Mullis in 1983. The amplified DNA can be cloned directly or used in a variety of analytical procedures.

The PCR procedure has an elegant simplicity. Two synthetic oligonucleotides are prepared, complementary to sequences on opposite strands of the target DNA at positions just beyond the ends of the segment to be amplified. The oligonucleotides serve as replication primers that can be extended by DNA polymerase. The 3 ends of the hybridized probes are oriented toward each other and positioned to prime DNA synthesis across the desired DNA segment (Fig. 9–16). (DNA polymerases

FIGURE 9–15 Specialized DNA libraries. (a)Cloning of cDNA next to a gene for green fluorescent protein (GFP) creates a reporter con struct. RNA transcription proceeds through the gene of interest (insert DNA) and the reporter gene, and the mRNA transcript is then ex pressed as a fusion protein. The GFP part of the protein is visible in the fluorescence microscope. The photograph shows a nematode worm containing a GFP fusion protein expressed only in the four “touch” neurons that run the length of its body. (b) If the cDNA is cloned next to a gene for an epitope tag, the resulting fusion protein can be precipitated by antibodies to the epitope. Any other proteins that interact with the tagged protein also precipitate, helping to elucidate protein-protein interactions.

FIGURE 9–16 Amplification of a DNA segment by the polymerase chain reaction. (a)The PCR procedure has three steps. DNA strands are 1 separated by heating, then 2 annealed to an excess of short synthetic DNA primers (blue) that flank the region to be amplified; 3new DNA is synthesized by polymerization. The three steps are repeated for 25 or 30 cycles. The thermostable DNA polymerase TaqI (from Thermus aquaticus, a bacterial species that grows in hot springs) is not denatured by the heating steps. (b)DNA amplified by PCR can be cloned. The primers can include noncomplementary ends that have a site for cleavage by a restriction endonuclease. Although these parts of the primers do not anneal to the target DNA, the PCR process incorporates them into the DNA that is amplified. Cleavage of the amplified fragments at these sites creates sticky ends, used in ligation of the amplified DNA to a cloning vector.

synthesize DNA strands from deoxyribonucleotides, using a DNA template, as described in Chapter 25.) Isolated DNA containing the segment to be amplified is heated briefly to denature it, and then cooled in the presence of a large excess of the synthetic oligonucleotide primers. The four deoxynucleoside triphosphates are then added, and the primed DNA segment is replicated selectively. The cycle of heating, cooling, and replication is repeated 25 or 30 times over a few hours in an automated process, amplifying the DNA segment flanked by the primers until it can be readily analyzed or cloned. PCR uses a heat-stable DNA polymerase, such as the Taq polymerase (derived from a bacterium that lives at 90 C), which remains active after every heating step and does not have to be replenished. Careful de sign of the primers used for PCR, such as including re striction endonuclease cleavage sites, can facilitate the subsequent cloning of the amplified DNA (Fig. 9–16b). This technology is highly sensitive: PCR can detect and amplify as little as one DNA molecule in almost any type of sample. Although DNA degrades over time (p. 293), PCR has allowed successful cloning of DNA from samples more than 40,000 years old. Investigators have used the technique to clone DNA fragments from the mummified remains of humans and extinct animals such as the woolly mammoth, creating the new fields of molecular archaeology and molecular paleontology. DNA from burial sites has been amplified by PCR and used to trace ancient human migrations. Epidemiologists can use PCR-enhanced DNA samples from human remains to trace the evolution of human pathogenic viruses. Thus, in addition to its usefulness for cloning DNA, PCR is a potent tool in forensic medicine (Box 9–1). It is also being used for detection of viral infections before they cause symptoms and for prenatal diagnosis of a wide ar ray of genetic diseases. The PCR method is also important in advancing the goal of whole genome sequencing. For example, the mapping of expressed sequence tags to particular chro mosomes often involves amplification of the EST by PCR, followed by hybridization of the amplified DNA to clones in an ordered library. Investigators found many other applications of PCR in the Human Genome Pro ject, to which we now turn.

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في ذكرى تأسيسه.. متحف الكفيل يستعرض مسيرته في حفظ التراث الإنساني والإسلامي

قسم الشؤون الفكرية وجامعة ميسان يبحثان تعزيز التعاون العلمي المشترك

قسم الآليات يوضح عمل شعبة السيطرة في خدمة الزائرين ودعم أقسام العتبة العباسية المقدسة

متحف الكفيل يعرض درهمًا فضيًّا يوثّق مرحلة ولاية العهد للإمام الرضا (عليه السلام)

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السجائر الإلكترونية والإقلاع عن التدخين.. نتائج متباينة تثير الجدل العلمي

مصر تحقق إنجازاً دولياً جديداً في منظمة الصحة العالمية

دراسة: السباحة فعّالة أكثر من الجري في الحفاظ على صحة القلب

عامل خطر في بداية الحمل يؤخر تطور الكلام والمهارات الحركية لدى الرضع

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علماء يكشفون سر سرعة الدلافين في السباحة

سلمي أو دموي.. هكذا تنتقل السلطة في مجتمع الجرذان

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مواضيع ذات صلة

From Genomes to Proteomes:- Detection of Protein-Protein Interactions Helps to Define Cellular and Molecular Function

From Genomes to Proteomes:- Cellular Expression Patterns Can Reveal the Cellular Function of a Gene

From Genomes to Proteomes:- Sequence or Structural Relationships Provide Information on Protein Function

From Genes to Genomes:- Genome Sequences Provide the Ultimate Genetic Libraries

From Genes to Genomes:- DNA Libraries Provide Specialized Catalogs of Genetic Information

DNA Cloning: The Basics:- Alterations in Cloned Genes Produce Modified Proteins

DNA Cloning: The Basics:- Expression of Cloned Genes Produces Large Quantities of Protein

DNA Cloning: The Basics:- Specific DNA Sequences Are Detectable by Hybridization

DNA Cloning: The Basics:- Cloning Vectors Allow Amplification of Inserted DNA Segments

DNA Cloning: The Basics:- Restriction Endonucleases and DNA Ligase Yield Recombinant DNA

Other Functions of Nucleotides:- Adenine Nucleotides Are Components of Many Enzymes Cofactors

Other Functions of Nucleotides:- Nucleotides Carry Chemical Energy in Cells