Deoxyribonucleotide triphosphates (dNTP) are essential components of the polymerase chain reaction (พีซีอาร์). dNTPs serve as the building blocks that allow DNA replication to occur. During พีซีอาร์, dNTPs enable the amplification of target DNA sequences. Understanding what dNTPs are and their function is key to appreciating PCR.
What Does dNTP Stand For?
dNTP stands for deoxyribonucleotide triphosphate. Deoxyribonucleotides are the monomers that makeup DNA. Each deoxyribonucleotide is composed of:
- A nitrogenous base: adenine (ก), thymine (ต), cytosine (ค), or guanine (G).
- A deoxyribose sugar molecule.
- A triphosphate group.
The ‘deoxy’ prefix indicates that the sugar component is deoxyribose rather than ribose. Deoxyribose lacks an oxygen atom on the 2′ carbon.
The triphosphate refers to the three phosphates attached to the 5′ carbon on the sugar. Having three phosphate groups makes dNTPs high-energy molecules.
How many types of dNTPs?
There are four different dNTPs, one for each of the DNA bases:
- ดีเอทีพี – deoxyadenosine triphosphate
- ดีทีพี –deoxythymidine triphosphate
- ดีซีทีพี –deoxycytidine triphosphate
- ดีจีทีพี –deoxyguanosine triphosphate
These four dNTPs are the basic building blocks needed for synthesizing new DNA strands.
What Are dNTPs in Biology?
In biology, dNTPs have a central role in DNA replication and PCR:
DNA Replication
During DNA replication inside cells, dNTPs are incorporated into new DNA strands as they are synthesized. The double-stranded DNA helix unwinds and splits into two single strands. Each strand acts as a template for a new complementary strand to be made.
DNA polymerase adds dNTPs that are complementary to the template strand, binding via base pairing. As the bases pair up, the sugar-phosphate backbones of the dNTPs are linked together.
This continues until both new strands are complete copies of the original DNA molecule. The dNTPs provide the necessary components to construct the new DNA strands.
Polymerase Chain Reaction
In PCR, dNTPs perform the same function as during DNA replication. PCR mimics cellular DNA replication in a test tube. The target DNA sequence is repeatedly copied using cycles of heating and cooling.
In each cycle, dNTPs are added by DNA polymerase to build new strands complementary to the separated template strands. This amplifies the target region exponentially. The dNTPs allow rapid synthesis of billions of copies of the DNA segment.
PCR could not proceed without dNTPs to polymerize new DNA strands.
How Do dNTPs Work in PCR?
dNTPs are an essential reagent that enables PCR amplification. Here is an overview of how dNTPs function in each step of PCR:
การเสียสภาพ
In the initial denaturation step, the double-stranded DNA is heated up to 94-98°C. Hydrogen bonds between the complementary strands break apart, yielding two single-stranded DNA molecules.
การหลอม
The reaction temperature is lowered to 50-65°C to allow primer annealing. Forward and reverse primers bind to complementary sequences flanking the target DNA region.
Extension/Elongation
In this step, DNA polymerase adds dNTPs to the primers, synthesizing new strands complementary to the template strands. The temperature is raised to 72°C, the ideal temperature for Taq polymerase enzyme activity.
Taq polymerase brings in the dNTPs to base pair with the exposed template bases. As each dNTP is added, a phosphodiester bond forms between it and the growing DNA strand.
This continues until the complete complementary sequence is formed. The result is two new double-stranded DNA copies containing the target region.
Repetition
The thermal cycling repeats – heating to separate the strands, cooling for primer annealing, and polymerase-mediated extension using dNTPs.
Each cycle doubles the number of DNA copies. After 30-40 รอบ, millions of copies of the target are made.
The dNTPs added in each extension phase enable the exponential amplification. Without dNTPs, new DNA strands could not be synthesized.
What Happens if dNTPs Are Left Out of PCR?
dNTPs are essential and required for PCR. If dNTPs are not added to the PCR master mix, the reaction will fail.
Without dNTPs, Taq polymerase has no nucleotides to add to the primers. ดังนั้น, the elongation of new DNA strands cannot occur. The target DNA will not be amplified at all.
Leaving out dNTPs means the PCR will not work, period. Only primers and template DNA will remain with no amplification.
A control reaction lacking dNTPs can be useful to confirm that DNA synthesis depends on dNTPs. But normally, the complete omission of dNTPs is an automatic failed PCR experiment.
Why Are ddNTPs Used in DNA Sequencing?
In DNA sequencing reactions, slightly modified deoxynucleotides called dideoxynucleotides (ddNTPs) are used. ddNTPs differ by containing a hydrogen atom on the 3′ carbon rather than a hydroxyl group.
This 3′ hydrogen prevents the formation of the next phosphodiester bond. When a ddNTP is incorporated by DNA polymerase, strand extension is terminated.
By using a ratio of dNTPs and ddNTPs, random termination occurs, generating DNA fragments of varying lengths. These can then be separated by size to reconstruct the original sequence.
ddNTPs allow the sequencing of DNA by abruptly halting synthesis at specific bases. Compared to normal dNTPs, ddNTPs lack the 3′ hydroxyl group needed to continue strand elongation.
What is the Typical dNTP Concentration in PCR?
The standard dNTP concentration in a PCR is generally 200 μM of each dNTP. This provides an optimal ratio of dNTPs to DNA templates for efficient amplification.
Too low of a dNTP level can lead to non-specific amplification and insufficient yields. Too high can reduce accuracy and increase nonspecific products.
200 μM per dNTP, รวมเป็น 800 μM dNTP, is suitable for most PCR applications. More or less can be used if needed to optimize the reaction.
For long PCRs, a higher dNTP concentration (500-1000 ไมโครเมตร) may improve yields for amplifying longer DNA targets.
It’s important to keep all four dNTPs present at equal concentrations. An unbalanced ratio can hinder the reaction.
บทสรุป
dNTPs are the nucleotide building blocks that allow PCR amplification to occur. These components include dATP, ดีทีพี, ดีซีทีพี, and dGTP.
In each PCR cycle, dNTPs are incorporated by Taq polymerase into new DNA strands complementary to the template strands. This enables exponential doubling of the target DNA region.
dNTPs are essential reagents for PCR. Without dNTPs, DNA polymerase cannot synthesize new copies of the target sequence. Amplification depends entirely on the presence of all four dNTPs.
Understanding the role of dNTPs helps us appreciate how PCR manages to produce billions of copies of DNA segments within hours. By providing the basic substrates for DNA replication, dNTPs sit at the core of this indispensable molecular biology technique.
