biology exam 3 ~ DNA Replication & PCR

replication

the biological process of copying a DNA molecule to produce two identical DNA molecules, ensuring each new cell receives a complete set of genetic instructions

origin of replication

where DNA replication is initiated (binding site for replication proteins/enzymes)

replication fork 

where two original parent strands open up & allow protein machinery to make new complimentary strand (two strands have been separated)

replication bubble

the region of DNA where the double helix unwinds and separates to allow for DNA replication to occur

helicase

enzyme that starts replication by breaking the hydrogen bonds between bases & allows the 2 DNA strands to separate

primase

sets down (RNA) primers on template strand (starts replication)

can be denovo or from nothing

DNA polymerase III

extends/synthesizes new DNA strand

reads template & makes compliment to build molecule

cannot start from nothing/has to make phosphodiester bond

DNA polymerase I

removes RNA primers & replaces with DNA

ligase

seals the backbone (gap) by making phosphodiester bonds

leading strand synthesis

continuously replicate until reaching end (new origin)

also known as continuous DNA replication

lagging strand synthesis

synthesized discontinuously in short (Okazaki) fragments

Okazaki fragments

short chunks of replicated DNA

Polymerase Chain Reaction (PCR)

a laboratory technique used to make millions to billions of copies of a specific segment of DNA from a very small sample

Taq DNA polymerase

a heat-stable enzyme, originally isolated from the bacterium Thermus aquaticus, that is crucial for polymerase chain reaction (PCR) because it can synthesize new DNA strands at the high temperatures required to separate DNA

It works by extending DNA primers using deoxynucleotide triphosphates (dNTPs) to create a new, complementary DNA strand.

dNTP’s

deoxyribonucleoside triphosphate, the building blocks of DNA. They consist of a deoxyribose sugar, a phosphate group, and one of four nitrogenous bases: adenine (dATP), cytosine (dCTP), guanine (dGTP), or thymine (dTTP)

primer

a short strand of RNA or DNA that acts as a starting point for new nucleic acid synthesis

denaturation step

breaks hydrogen bonds between bases (initial stage) 95 degrees celcius

annealing step

where the reaction temperature is lowered (55) to allow short DNA primers to bind to their complementary sequences on the single-stranded template DNA

extension step

a stage where DNA polymerase synthesizes a new DNA strand by adding nucleotides to the 3' end of each primer, creating a complementary copy of the target DNA sequence (72/68)

results in amplification of DNA

amplicon

size of DNA fragments

actin

a globular protein that polymerizes to form microfilaments, which are crucial components of the cell's cytoskeleton in eukaryotic cells

“master mix”

includes the buffer, enzyme, and dNTPs in a single solution