Essentials of Genetics

Unit 4: How Do Scientists Study and Manipulate the DNA inside Cells?

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DNA Is a Structure That Encodes Biological Information

Discovery of the Function of DNA Resulted from the Work of Multiple Scientists

The Information in DNA Is Decoded by Transcription

The Information in DNA Determines Cellular Function via Translation

Introduction: How Does DNA Move from Cell to Cell?

Replication and Distribution of DNA during Mitosis

Replication and Distribution of DNA during Meiosis

DNA Is Constantly Changing through the Process of Recombination

DNA Is Constantly Changing through the Process of Mutation

Some Sections of DNA Do Not Determine Traits, but Affect the Process of Transcription: Gene Regulation

Introduction: How Is Genetic Information Passed between Organisms?

Each Organism's Traits Are Inherited from a Parent through Transmission of DNA

Inheritance of Traits by Offspring Follows Predictable Rules

Some Genes Are Transmitted to Offspring in Groups via the Phenomenon of Gene Linkage

The Sex of Offspring Is Determined by Particular Chromosomes

Some Organisms Transmit Genetic Material to Offspring without Cell Division

Introduction: How Do We Study the DNA Inside Cells?

The Order of Nucleotides in a Gene Is Revealed by DNA Sequencing

Scientists Can Make Copies of a Gene through PCR

Scientists Can Analyze Gene Function by Deleting Gene Sequences

Gene Expression Is Analyzed by Tracking RNA

Scientists Can Study an Organism's Entire Genome with Microarray Analysis

Introduction: How Does Inheritance Operate at the Level of Whole Populations?

The Collective Set of Alleles in a Population Is Its Gene Pool

The Variety of Genes in the Gene Pool Can Be Quantified within a Population

The Genetic Variation in a Population Is Caused by Multiple Factors

Genomics Enables Scientists to Study Genetic Variability in Human Populations

4.3 Scientists Can Make Copies of a Gene through PCR

Once scientists have zeroed in on a specific segment of DNA, how do they produce enough copies of that segment for their research? In most cases, the polymerase chain reaction, or PCR, is their method of choice for quickly generating a sufficient amount of identical genetic material for study and analysis.

Prior to the development of PCR in the 1980s, the primary method for producing many copies of a gene was a relatively time-consuming process known as DNA cloning. This technique involved insertion of the gene of interest into living bacterial cells, which in turn replicated the gene along with their own DNA during the division and replication processes.

What is PCR?

The key element of PCR is heat. Throughout the PCR process, DNA is subjected to repeated heating and cooling cycles during which important chemical reactions occur. During these thermal cycles, DNA primers bind to the target DNA sequence, enabling DNA polymerases to assemble copies of the target sequence in large quantities.

PCR makes it possible to produce millions of copies of a DNA sequence in a test tube in just a few hours, even with a very small initial amount of DNA. Since its introduction, PCR has revolutionized molecular biology, and it has become an essential tool for biologists, physicians, and anyone else who works with DNA.

How does PCR work?

Figure 1: The various components required for PCR include a DNA sample, DNA primers, free nucleotides called ddNTPs, and DNA polymerase.

PCR relies on several key chemical components (Figure 1):

A small amount of DNA that serves as the initial template or target sequence
A pair of primers designed to bind to each end of the target sequence
A DNA polymerase
Four dNTPs (i.e., dATP, dCTP, dGTP, dTTP)
A few essential ions and salts

The PCR process then uses these ingredients to mimic the natural DNA replication process that occurs in cells. To automate this process, a machine called a thermocycler jump-starts each stage of the reaction by raising and lowering the temperature of the chemical components at specific times and for a preset number of cycles.

Each cycle of PCR has three main steps, as described in the following sections.

Step 1: Denaturation

Figure 2: When heated, the DNA strands separate.

During the first step in PCR, the starting solution is heated to the necessary temperature, usually between 90^° and 100^°C. As the heat builds, it breaks the bonds joining the two strands of the DNA double helix, thereby enabling the DNA to separate into two single strands. This "melting" of the DNA into single strands is called denaturation (Figure 2).

Step 2: Annealing

Figure 3: When the solution is cooled, the primers anneal.

After it is held for several minutes at the initial target temperature, the reaction mixture is quickly cooled, usually to between 30^° and 65^°C. The mixture is then held for less than one minute at this temperature. This gives the primers an opportunity to bind, or anneal, to their complementary sequences on the single strands of DNA (Figure 3).

Step 3: Extension

Figure 4: DNA polymerase attaches to each primer and assembles dNTPs to build a new strand.

During the final, or extension, stage of PCR, the sample is heated again, usually to between 60^°and 75^°C, and it is held at that temperature for less than one minute. At this point, the DNA polymerase begins making a new DNA strand by attaching to the primers and then adding dNTPs to the template strand, thereby creating a complementary copy of the target sequence (Figure 4).

Figure 5: The replication cycle repeats many times.

The number of new copies of the DNA sequence of interest doubles with each three-step cycle. Thus, if the PCR process is repeated 40 or 50 times, even small samples of template DNA can yield millions of identical copies (Figure 5).

PCR is an incredibly versatile technique with many practical applications. Once PCR cycling is complete, the copied DNA molecules can be used for cloning, sequencing, mapping mutations, or studying gene expression.

Variations on conventional PCR

Recently, PCR has proven useful in ways beyond merely copying and propagating identical segments of DNA. Today, geneticists rely on PCR to aid in the study of genes themselves.

Copying and quantifying DNA at the same time using real-time PCR

One modification of conventional PCR allows researchers to copy a particular DNA sequence and quantify it simultaneously. Dubbed quantitative real-time PCR (qPCR), this technique makes it possible to measure the amount of DNA produced during each PCR cycle. This refinement involves the use of fluorescent dyes or probes that label double-stranded DNA molecules. These fluorescent markers bind to the new DNA copies as they accumulate, making "real-time" monitoring of DNA production possible.

As the number of gene copies increases with each PCR cycle, the fluorescent signal becomes more intense. Plotting fluorescence against cycle number and comparing the results to a standard curve (produced by real-time PCR of known amounts of DNA) enables scientists to determine the amount of DNA present during each step of the PCR reaction.

Quantifying RNA using reverse transcription PCR

More about gene copying

Real-time PCR can also be used to calculate the amount of specific kinds of genetic material other than DNA, such as RNA. This extension of real-time PCR technology, called reverse transcription PCR (RT-PCR), combines real-time PCR with reverse transcription, the process that makes DNA from mRNA. RT-PCR can be used to determine how gene expression changes over time or under different conditions. For this reason, this technique is sometimes used to verify microarray data.