Chapter 14

• replication of DNA • locating nucleotide sequences • cutting and joining sections of DNA • seperating pieces of DNA • pasting DNA into an organism • sequencing sections of DNA

polymerse chain reaction (PCR) under normal conditions a nuber of enzymes are required for replication PCR uses one enzyme: a heat resistance form of DNA polymerse

organisms with enzymes that can withstand high temps

sperate strands of DNA hydrogen bond - heat to 90 C- single strand DNA - cool double stranded DNA

short DNA strands - required to start copying

synthesizes new strands from unpolymerized nucleotides

sample DNA, primers, DNA polymerse, and nucelotides are combined 1. heat strands to sperate 2. cool to allow binding of primer and sythesis of new strands 3. repeat each step doubles the amount of DNA

restriction enzymes: cut DNA into fragments naturally found in bacteria probably used by to cut viral DNA in techological applications, DNA samples must be removed from a cell

ech enzyme cuts DNA at specific nucleotide sequences cut is not directly across strand - leaving sticky ends complimentary sticky ends form hydrogen bonds

DNA ligase: bonds the phosphate of one nucleotide to the sugar of another nueclotide

uses a RNA sequence to find and cut DNA can cut DNA at nay nucleotide sequence can be introduced to a livng cell to make a DNA cut

cut with the SAME restiction enzyme

• DNA from each organism must be cut with the same restiction enzymes • DNA from each organism is mixed, allowing H bonds to form between sticky ends • DNA ligase bonds sugar/ phosphate backbones

3 methods: only covering 1 plasmids 2. virususes ( not covered) 3. mechanical methods (not covered)

plasmids have same restrictins sites as those found on chromosomal DNA bacteria naturally take up plasmids from the environment soem plasmids can intergrate into eukaryotic genomes

placing human genes into bacteria to produce human protiens • extract plamid from bactria and DNA from human • cut with same restriction emzymes • mix samples, add ligase • place plasmid back in bactria. select cells that produce gene product

plasmid have a gene that allows it to become part of a plant chromosome

gel electrophoresis DNA has a neg charge and can therefor be drawn to a pos charge small DNA fragments move more quickly to pos charge than larger fragments

• mixure of DNA placed on gel • electric current is applied cross the gel • DNA sperates by size • DNA is stained uses: indentify organisms sperating and locating genes sequencing DNA

sequencing uses of PCR and two types of nueclotides • normal nucleotides • modified nucleotides: - different dyes are attached to nucleotides with different bases - sugars cannot bind to a phosphate (stops PCR)

probes: known sequences of single stranded, radioactive or fluorescent DNA

• sample DNA is seperated into single stands and attached to the surface • probe is added to the sample probe binds iwth comlimentry strands • sample washed to remove unbound probes • prescence of probe detected

• detect a sequence in an organism: - allele of interest (known mutation) - presence of a mirco organism in diease idenification • genetic mapping. combined with gel electrophoresis

examines size distrubution of DNA fragements (restriction fragments) cut by restiction enzymes size of fragments is determined by distance between restriction sites