Chapter 6 - PCR Bio-Rad

polymerase chain reaction

in vitro (outside of cell), targeted for a specific sequence, done to amplify DNA quantity

Template DNA, Forward and reverse primers, heat stable DNA polymerase, dNTPs, Buffer, MgCl2

dNTPs + Buffer + MgCl2

PCR is very sensitive to contamination

short pieces of DNA that mark the beginning and end of the target DNA

This is a factor that effects the PCR reaction. Ideally, primers in the pair must be within a few degrees of each other.

builds DNA by adding nucleotides (original one from Thermus Aquaticus)

doubles each cycle, exponential

denaturation, annealing, extension

(~94 C) breaks hydrogen bonds holding double stranded DNA together to produce single stranded DNA

(40-60 C) cools down to allow primers to hydrogen bond to the DNA sequence

(~72 C) warm temperature to optimize the rate that the DNA polymerase adds nucleotides to the DNA strand, template strand reads 5' -> 3' while new strand reads 3' -> 5'

grind up and heat food sample to 95 C to release DNA, use InstaGene Matrix to remove metal ions that the DNA needs to function (protects DNA from degrading and is more efficient and faster)

multiple primers for multiple targets

newly made DNA is tagged with a fluorescent dye; the levels of fluorescence can be measured after every PCR cycle

small amount of dNTPs are ddNTPs, missing one more oxygen to prevent the chain from growing further

Portions of the DNA that are extremely variable between people, analyzing STRs will greatly reduce the chance of two people matching

A sequence of DNA repeated at different locations on different chromosomes that are only in primates, repetitions caused by sequence copying onto mRNA and then reinserting into genome with reverse transcriptase

PCR products

using genetically modified animals to make pharmaceutical drugs

sugar, phosphate, base

is used to copy and amplify minute quantities of DNA/

bacterium that can sustain high temperatures without being denatured

A heat-stable form of DNA polymerase extracted from bacteria that live in hot environments, such as hot springs, that is used during PCR technique

process that takes place in a living organism

small tube that holds .2mL

2nd set of primers used just inside of first set

process of using agents to "clean" DNA sample and remove other cellular proteins

used to study expression of numerous genes