Chapter 14 Flashcards
6 basic methods
- 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
replication 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
hot springs contain
organisms with enzymes that can withstand high temps
heat is used to
sperate strands of DNA
hydrogen bond - heat to 90 C- single strand DNA - cool double stranded DNA
primers
short DNA strands - required to start copying
heat resistance DNA polymerse
synthesizes new strands from unpolymerized nucleotides
steps in PCR
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
cutting and joining 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
restiction sites
ech enzyme cuts DNA at specific nucleotide sequences
cut is not directly across strand - leaving sticky ends
complimentary sticky ends form hydrogen bonds
joining sections of DNA
DNA ligase: bonds the phosphate of one nucleotide to the sugar of another nueclotide
NEW* the cas 9 emzyme
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
combining DNA from different organisms
cut with the SAME restiction enzyme
combining DNA from different organisms
- 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
pasting DNA into organisms
3 methods: only covering 1
plasmids
2. virususes ( not covered)
3. mechanical methods (not covered)
plasmid vectors
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
plasmid vectors ex
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
plamid introduced into cultured plant cells
plasmid have a gene that allows it to become part of a plant chromosome
separating pieces of DNA
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
procedure gel electrophoresis
- 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
determining the nucelotide sequence of a sction of 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)
locating nucleotide sequences
probes: known sequences of single stranded, radioactive or fluorescent DNA
locating sequences steps:
- 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
uses for probes
- 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
restriction fragment anaylsis
examines size distrubution of DNA fragements (restriction fragments) cut by restiction enzymes
size of fragments is determined by distance between restriction sites
mutations in restiction sites create
different size DNA fragaments (bands)
uses
- identify carriers of alleles associated geentic disorders (especially recessive alleles)
- DNA fingerpriting: indentify indivduals within a population
restriction fragement anaylsis ex)
mutation that causes sickle cell anemia can be identified with restiction enzyme
this only works if the mutation resulting in a new alleles is part of a restiction site
human DNA fingerprinting
examines regions of DNA that areL
non coding
highly variable
repeated
doesnt rely on mutations to restiction sites
allele- # of repeats (a different size of DNA fragment)
average number of alleles in the population is uusally about 30
throughout the chrosmosome there are many locations with repeats
each location with a repeat can be examined using restiction fragment anaylsis
human DNA fingerprinting procedure
uses restiction fragments and probes
- DNA sample is amplified using PCR
- sample is cut with restirction enzymes
- fragments are sperated by gel electrophoresis
- DNA seprated into single strands and bound to a surface
- probes are used to find fragments with repeated sequence
testing identity
to test for the identity of indivduals, more than one location of repeated DNA is compared between samples
parental testing
bands (alleles) found in a child must be present in one or both parents
if the mother doesnt havea band the child posses, it must be in the father
genetically modified organisms (GMOs)
organisms that have had their genomes artifically modified
2 types of mofications
modification of exisiting genomes
trangenic organisms
modification of existing genomes
ex) flavor savr tomatoes
have a gene responsible for fruit softening silenced
transgenic organisms
organisms with genes from two or more species
ex) 1. round up ready crops:
have a gene from bacteria making them resistant to “round up”
2. Bt crops:
genes from bacteria make them toxic to insects
using genetic technology to treat genetic disorders
- supply missing gene product
working copies of genes inserted into other organisms to produce gene product
ex) human insulin is produced from E.coli bacteria
gene therapy
place normal in a vector
infect cells
normal gene becomes part of cells chromosome
inject modified cells back into patient
extract cells from patient with abnormal allele
