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DNA3 Types PCR/SNPS
Types of PCR and SNPs.
| Question | Answer |
|---|---|
| What types of PCR is there? | Standard,Multiplex,Temperature Gradient PCR,Touchdown PCR,Hot Start PCR,Nested PCR, Rapid PCR, Real Time PCR. |
| Why have so many types of PCRs? | Modified and alternative PCR protocols are useful if something goes wrong or for optimizing PCR reactions. |
| What is Temperature Gradient PCR? | Gradient of Temperatures spread out. Often the annealing temperature is significantly higher than the one based on the primer T. Tm can be influenced by components of a PCR reaction. What is Tm? Standard Temp= 5 c below Tm of primer. |
| Temperature Gradient PCR info/problems? | Not having the optional annealing temp for primers can result in having secondary bands. |
| Annealing temp info? | Higher annealing temp will minimize non specific binding. A lower temperature means things move slower. If a portion of the primer complements the DNA it could prime and elongate.(resulting in non specific product) |
| Touchdown PCR? | Same idea as Temperature gradient PCR.(truing to eliminate non specific products that swap out product) Touchdown starts with a high temperature. Slowly drops tempature after first few cycles. |
| More Touch Down? | High start temp ensures that only correct PCR products are amplified. (provides extra copies of DNA template for subsequent cycles.) Ensure that the correct PCR product beats out non specific products. |
| What is Hot Start PCR? | Deals with Taq being active at low temps. Combats by starting reaction at higher temp. (introduce DNA polymerase after a needed temp has been reached.) |
| What is Nested PCR? | The goal is to avoid non specific PCR products from misprinting. Uses two sets of reaction. One on top of the nest. (amplify one and then using the product of first reaction you run a more targeted primer set on top of the first one) |
| What is Rapid PCR? | Same as regular PCR however the length of each cycle is reduced dramatically. The result is improved quality of reaction products because of more precise primer-template annealing. Misprinting does not happen because of high temps. |
| What is Real Time qPCR? | Allows you to detect and quantify your product simultaneously. Quantity can be absolute number or abundance when compared to reference DNA or input. Real Time PCR happens by using non specific florescent dyes that interchelate. |
| Explain how real time detection occurs? | Happens because DNA probes which have oligonucleotides that are labeled with a florescent reporter which permits detection after hybridization of the probe with the target DNA. |
| SNPs | Single Nucleotide Polymorphism. Results from a single base variation between points in the genome. Abundant in the human genome (millions) SNPS are bi allecic and have 3 possible phenotypes. (AA,BB,AB) |
| Advantages of SNPs | PCR products can be less than 100 bp in size. Can be multiplexed to a higher level than STRs because some detection methods are not constrained by electrophoresis. No stutter affects. Can pick out eithnic and other physical markers. |
| Disadvantages of SNPs. | Less discriminatory than STRs because they are bi allelic. 50-100 SNPs are needed to get the same power of discrimination versus 10-16 STR loci. Sample mixtures are an issue.(as with most methods) Do you have a mixed samples versus a heterozygous |
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