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cutting DNA into specific pieces and rearranging them as desired for a particular purpose
- "restruction endonucleases"
- Enzymes that out @ particular sequence in middle of DNA (from bacteria)
- -Recognize certain sequence (4-12 nucleotides long)
- Symetrical- "Pallindrome" eg hannah
- Sticky ends (same on both ends)
Frog Dna + Bacteria DNA
- Frog Dna + Bacteria DNA (plasmids) = sticky ends combine to make frog protein
Carriers of DNA into organism:
- 1) plasmid - small round piece of dna
- 2) animal virus
- 3) bacteriaphage - virus that attacks a bacterium
- methode of seperating mixture of molecules
- DNA, protein, mRNA, fragments/pieces
- size and or charge
- Uses gel
Four stages of experiement:
- 1.) cut the dna from source. cut from reciprocol dna
- -use restricton endonuclease enzyme on -
- cut out:
- a) animal dna that has the gene of interest
- b) bacterial dna (plasmid)
2.)Mix dna sources (animal and bacteria) - stick together (sticky ends) - recombinant dna
3.) Insert bacteria plasmids back inside bacteria
- 4.) screening for bacterium w gene of interest "hybridization" probe radioactive single stranded dna of gene of interest
- -apply solution that denatures dna *single stranded
- -add probe
- -probe sticks to gene of interest
- used to quicken the proc. described above
- -extract mRNA from a cell already making it
- mRNA ----> cDNA c=complimentary
- ---> = reverse transcenphase
- Start w/ mature mRNA make gene (cDNA) ---> put into bacterium---> make product
- *much faster, no screening
- Polymerase chain reaction:
- Make many copies of DNA fragment rapidly
- Dna sample
- Dna polymerase *Thermostable
- Short DNA primer
- Thermal cycler (instrument)
Stages of PCR:
- 1.) Denaturation *end up w single strands
- 2.) Annealing (attachment)
- 3.) Synthesis of DNA
- Hot to cold to medium, repeat
- Add primer, makes 2 copies, into 4, into 8.....
Southern Blotting and RFLP Analysis
- Old technique for DNA fingerprinting
- 1.) cut dna into fragments under restriction endo nuclease
- population is polymorphic w respect to cutting sites (variable)
- RFLP - Restriction Fragment Length Polymorphism
- 2.) seperate fragments using gel electrophorsis
- Gel - seperated onto paper (single stranded) - use probe to match DNA
How is PCR used?
- 1.) dna fingerprinting
- -replaced RFLP based technigue in US
- New form of variabilty used are # of STRS
- STR: short tandem repeat
- We are all variable *polymorphic in # of STRS
- STR - 3-5 Base pairs long
- Single Tandem Repeat
- 3-5 base pairs long
- ex: THO1 locus on chromosome 11 TCAT (1-20 times in us)
- 13 Loci used
- Ex: locus 1 - crime scene dna alleles 6-3
- suspect A - alleles 6-3
- (3% of pop. have 6-3 alleles)
- Locus 2 - crime scene dna has alleles 10-15
- Suspect A - alleles 10-15
- 5 % of pop has this combo
- When we have 3% annd 5% - 1/650 match.
- *need to compare 13 diff loci
Identification of disease carriers (heterozygous)
- One procedure - amplify DNA w/ PCR
- -use restricition enzyme to make fragments
- -seperate fragments using gel elelctrophosis
- PCR based
- amplify 16 loci (each is str site)
- seperate using gle electroph.
- Compare loci and match (1 from mom, 1 from dad)
DNA sequencing value:
- 1) medical and forensic application:
- a) medical - if you know what nucletotide seq. then you may know a.a. seq. then you may know form of protein, may know fuction, may know mutation and how it changes protein, can determine what drugs to help.
- b) forensic - SNP's (single nucleotide pholymorphisms)
- -scan DNA and identfy individual
- 2.) scientific value: commpartative genomics
- -some genes in yeast compare in humans.
- 1.) Diagnosis of diseases
- a) identify carrier of a disease
- b) using PCR to amplify foreing DNA in a cell - recognize foreign DNA.
- 2) human gene therapy - replacement in an individual of non functioning gene w/ a good one.
- 3.) pharmaceutical products-
- Ex. insulin, human growth hormone, TPA
Agricultural applications (plant):
- Agrobacterium (tumefaciens) = vector (carries gene)
- plasmid- into plant cell and causes infection
- 1. herbicid resistance "round up"
- herb res. gene---> cotton, corn, soy beans....
- 2. Increase nutritional value of crop
- 3. Nitrogen fixation - process gaseouis nitrogen (nitrogen fixing bacteria) Transfered to organic form
- 4. Biopharming - making plants produce pharmaceuticals
Animal apllications (spider goat)
- Should we worry about genetically modified food (GTM)
- 1. environment - local plants that are related to GM product.
- 2. Should consumers always be told.
- 3. Could consumers be hurt by GM food?
complimentary of eachother
cleave dna / like a knife
dna in a vector - complex mixture
entire genome in a vector
- southern: DNA
- norther: mRNA
- western: protein