> See also: > - [[DNA Polymerases]] > - [[DNA Hybridization]] > - [[Nucleic Acids]] # Polymerase Chain Reaction (PCR) PCR is a biotechnology technique for amplifying strands of genetic material within a sample. PCR uses the basic principles of [[DNA Replication|DNA replication]] An **amplicon** is a DNA or RNA fragment that has been amplified or copied multiple times through PCR. ## Procedure 1. *Denaturation:* Splitting dsDNA into ssDNA at high temperature - Typically (~94$\degree C$) 2. *Annealing:* Attaching primer to each piece of ssDNA - Varies depending on primers used, typically ~60 $\degree C$ 1. *Extending:* Elongation of primers by DNA polymerase at 3\` end, creating new dsDNA The PCR reaction is actually going towards equilibrium as the dNTPs are incorporated into the new strands after each round of extension. It’s important to understand that both directions of the of new synthesis are not being performed at the same time. Whenever one DNA polymerase begins, it’s starting position - [ ] How is there enough physical space for the polymerase enzyme to both latch onto the strand and immediately begin adding nucleotides? - I feel like a lot of enzymes we’ve studied need a sort of landing platform - It kind of does need a platform, and that platform seems to be the primers used in the reaction. Keep in mind that the primer strands and the amplicon strands are not the same. The primer has to hybridize **Primer Dimers** ![[Pasted image 20240915155002.png|325]] ### Components/Reagents > [!summary] Primer Design > Two short strands of DNA known as **primers** must be created to specify the start and end points of where the PCR will occur. A forward and reverse primer must be designed based on the target DNA sequences being amplified --- *Taq Polymerase* - Contains Polymerase Domain (Base Adding Section) - Contains 5'→3' Exonuclease Domain ### Thermocyclers **Thermocyclers** are machines that can automatically transition between temperatures based on a given set of instructions (duration & temperature). > [!important] **Calculating Melting Temperature** > $T_m=2(A+T)+4(G+C)$ > - Both the forward and reverse primers should be designed to have a similar melting temperature ($T_m$) > - The annealing temperature should always be ~5$\degree C$ lower than the lowest of the $T_m$ values calculated for each primer ### Clean-Up After a PCR is complete, the sample will likely contain *impurities* such as the primers and DNA polymerases The DNA can be run on an agarose gel to separate the target DNA from both debris and unwanted DNA/RNA strands ## Types of PCR | Name | Description | | --- | --- | | RT-qPCR | Quantifies RNA in a sample in real time | | TA Cloning | Ligating PCR products with A overhangs directly into vectors | | Nested PCR | Increases PCR's specificity using internal primers | | Multiplex PCR | Uses multiple primers to amplify several targets | | AFLP PCR | Amplifies DNA from RFLP adapters | | RAPD PCR | Amplifies DNA from random pairs | | Name | Description | | --- | --- | | Asymmetric PCR | Generates probes using a limiting primer | | Anchored PCR | Used to amplify DNA when the 3' end is unknown | | Inverse PCR | Inverts DNA to amplify upstream/downstream sequences | | Isothermal Amplification | PCR at room temperature, without the thermocycler | | Aptamers | Short DNA oligos that bind specifically to a target | ### Quantitative PCR (qPCR) > *Also referred to as "Real-Time PCR"*, however, because another technique called "Reverse-Transcriptase PCR" exists that name can be confusing. One major downside of traditional PCR is that the success of the experiment can only be determined afterward by running the sample on an agarose gel. **Quantitative Polymerase Chain Reaction (qPCR)** measures the kinetics of the reaction in the early phases of PCR, providing that a distinct advantage over traditional PCR detection. **SYBR Green Dye** is a nonspecific alternative to reporter probes ### Overlap-Extension PCR ### Reverse Transcription PCR (RT-PCR) Combines the reverse transcription of RNA into DNA and the amplifiication of it through PCR