Chapter 6 - PCR Bio-Rad

PCR

PCR

polymerase chain reaction

What is PCR?

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

What do you need for PCR?

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

Master Mix

dNTPs + Buffer + MgCl2

Why do you need aerosol tips for PCR?

PCR is very sensitive to contamination

Primers

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

melting temperature (Tm)

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

DNA polymerase

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

DNA in PCR

doubles each cycle, exponential

PCR steps

denaturation, annealing, extension

Denature DNA

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

annealing

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

Extension of DNA

(~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'

extracting DNA from food for PCR

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)

Multiplex PCR

multiple primers for multiple targets

Real Time PCR

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

PCR sequencing

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

STRs

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

Alu insertion

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

Amplicons

PCR products

Pharming

using genetically modified animals to make pharmaceutical drugs

Nucleotides are made up of

sugar, phosphate, base

PCR (polymerase chain reaction)

is used to copy and amplify minute quantities of DNA/

Thermus aquaticus

bacterium that can sustain high temperatures without being denatured

Taq polymerase

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

In Vivo

process that takes place in a living organism

PCR tube

small tube that holds .2mL

Nested PCR

2nd set of primers used just inside of first set

chelating

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

Microarrays (gene chips)

used to study expression of numerous genes