What is PCR amplification?
PCR stands for “Polymerase Chain Reaction”. This is a molecular biology technique used to copy and exponentially amplify specific sequences of DNA. You’ll learn more about it next week.
Can PCR be used to fuse a gene with a promoter or terminator sequence? If so, how likely is it to succeed?
In a word, yes. PCR is an important tool in the molecular cloning toolbox and can be used to help combine endless combinations of sequences. However, I think the concepts you are getting at here are “reading frame” and “fusion protein”. Proteins are modular and their component features can be combined to make new proteins with their composite functions. When you are creating a new gene that codes for a fusion protein, each component of the protein that will be translated needs to be in frame with the start codon. Upstream of the start codon, the promoter can tolerate a few extra bases between the end of the promoter region and the start codon (RNA polymerase will “search” for the first ATG). Likewise, downstream of the stop codon, the terminator sequence is important for release of the transcriptional complex (it’s not particularly relevant for translation). https://blog.addgene.org/plasmids-101-terminators-and-polya-signals
What is the difference between PCR and Sanger sequencing?
PCR uses 2 primers and amplifies a single product until it dNTPs become rate limiting; amplification is ceased at the plateau of the exponential phase. Sanger uses a repeated cycles of a polymerase reaction from a single primer. During amplification, the ddNTPs will randomly sit on the DNA template and terminate the extension. The dNTPs sit on the remaining templates and continue extending. Multiple cycles are performed to ensure that there are detectable amounts of ddNTP terminated sequences for each base in the original template; amplification is ceased within the linear phase of PCR. https://agctsequencing.wordpress.com/2012/02/10/sanger-sequencing-amplification-compared-to-basic-pcr/
I’m confused – how do primers work?
Primers work by the power of hydrogen bonds. Primers are just short sequences of single-stranded DNA. As long as a DNA sequence is relatively short, we (companies, really) can synthesize it from scratch without having to clone it into a cellular host. You can purchase a lifetime supply of a 20-mer for ~$5 from IDT. In a PCR, we specifically select the primer sequence so that it is complementary to either the 5′ or 3′ end of the stretch of DNA that we want to amplify. The denaturation step turns double-stranded template DNA into single-strands of DNA, which exposes A/T/G/C base pairs. The atomic structures of A/T and G/C allow for hydrogen bonds (2 and 3 of them for A/T and G/C pairs, respectively) to form. These hydrogen bonds are actually all that holds DNA together in the double-stranded form. So, when a single-stranded primer and a single-stranded template are complementary and form hydrogen bonds together, this creates a stretch of double-stranded DNA that is as many base pairs long as the primer. In PCR, DNA polymerase requires these dsDNA priming points to start filling in complementary nucleotides outside of the primer sequence. If you’re still confused, check out the re-booted PCR and primer design videos, and if that doesn’t clarify things, please make an office hours appointment with me.
