DNA Technology Ch 13

I. Vectors =Things use to transport genes into cells

A. Plasmid Vectors  (Plasmid = small circle of bacterial DNA)  1. contain only a few genes (non- essential )  2. bacteria will take in plasmids from the environment  3. only bacteria will take in plasmids

B. Virus Vectors  1.Viruses inject DNA into nucleus of host cell  2.We replace virus DNA with genes we want placed in a cell  3.Used in gene therapy  4.Treated ‘B in B’ disorder  a. virus put new gene in stem cells  b. stem cells returned to bone marrow  c.10 of 11 cured (3 later got leukemia)

II. Restriction Enzymes  A. Enzymes that cut DNA ONLY at specific sites  B. Sites are identified by nucleotide patterns on both strand of DNA  C. So only cuts double stranded DNA  D. Restriction enzymes were isolated from Bacteria  E. Bacteria use them as primitive immune system to cut virus DNA

III. cDNA = complimentary DNA  A. Why cDNA?  1) used to put human gene in bacteria  2) bacteria can’t remove introns from mRNA transcripts  3) we remove introns from the gene before we give it to them  4) we let the human cell do transcript processing  5) then use mRNA as a template

B. Making cDNA  1. Get human mRNA for needed protein  2. Add reverse transcriptase that bonds DNA nucleotides to RNA  this DNA is the cDNA  3.Add DNA polymerase to remove RNA nucleotides and replaces them with DNA nucleotides  4. End result = double stranded DNA  can be cut with restriction enzymes  & added to plasmids that bact. take in. 

IV.Probe used to locate gene of interest  A. Short strand of DNA complementary to gene of interest  B. Tagged with radiolabel or tracer  C. Binds to gene of interest to make hybrid DNA  D. Hybrid DNA = any DNA where the 2 sides of the double helix come from different sources

V.DNA Isolation – isolate the gene you want from the rest of the genome  A. Use probes to label DNA  B. Cut DNA using restriction enzymes  C. Separate DNA using electrophoresis  D. Use only DNA that includes the radiolabeled probe  E.Reduces volume of unwanted DNA

VI. Primer =  A. Short, synthetic, single strand of RNA or DNA  B. Complimentary to DNA in front of GoI  C. Initiates DNA synthesis

VII. PCR = polymerase chain reaction  Step A Mix DNA containing GoI with  1) primers for target gene  2) spare nucleotides (A, T, G, C)  3) DNA Polymerase from Thermus aquaticus bacteria….called Taq polymerase……heat resistant enzyme

 Step B : heat DNA strands to break H-bonds between base pairs and separate the strands  Step C: cool DNA so base pairs re- bond ( some bond to primer)  Step D: repeat  

VIII. Gel Electrophoresis- separates DNA based on size of fragment  A. Multiple samples of DNA  B. DNA cut with same restriction endonucleases  C. Dye added to visualize DNA  D. Samples placed in wells of gel (indentations)  E. Gel placed in liquid buffer

 F. Electric current run through buffer & gel  G. DNA migrates through the gel to positive end  H. Larger pieces have trouble moving through so move slower

I. Factors affecting DNA movement  1. More porous gel speeds up movement  2. Higher current speeds up movement

IX. Gel analysis  A. DNA standard marker of known length are in lane 1  B. Measure distance each segment moved  C. Graph distance vs size  D.Best fit line  E. Estimate unknown size

X. DNA Fingerprinting  Human genes 99% the same  Differences are in non-coding DNA between genes  Tandem Repeats = 2 bases repeated  ATATATAT # repeats varies so  Different people have different lengths of DNA between genes

DNA Fingerprinting Uses  Crime scene investigations  Paternity tests  Cut all samples with same enzymes  Do PCR on Samples to amplify DNA  Do electrophoresis to sort DNA by size  Compare results to crime scene or potential parents  1 in 3,000,000,000,000 chance of unrelated people sharing same DNA fingerprint

 Child gets all DNA from Mom or Dad  Any fragment found in child must be in either Mom’s DNA or Dad’s DNA

DNA Sequencing  Alter nucleotides so they will stop replication  Add modified A nucleotides to one sample  T to second, G to third, C to fourth  Run PCR then electrophoresis  Order of segments indicates order of bases in sequence

Sanger Method (dideoxy method)  Uses Dideoxynucleotide to stop replication  named after Frederick Sanger,  won the 1980  Nobel prize  in chemistry 

DNA Sequencing  Label Nucleotides with fluorescent dye each base different color  Alter labeled nucleotides so they stop DNA replication  Add both labeled nucleotides and normal nucleotides to sample DNA  Run PCR and electrophoresis

 Segments separated by size  Base that fluoresces is always the last base in line

Genetic Engineering  A) GMO – genetically modified organism  1) foreign genes added to organisms  2) bt gene from Bacillus thuringiensis  a) bacterial protein toxic to insects  b) used by WHO to kill mosquitoes  c) used by organic farmers  d) added to corn potato cotton so plant makes its own bt protein insecticide  e) specific to borers & bollworm 

 3) bt mechanism  a) plant uses bt gene to make protein  b) insect eats protein  c) protein binds to receptors in gut  d) gut wall breaks down  e) only insects with receptor affected  f) studies show no harm to Monarchs   g) advantages  1) less pesticide  2) more specific delivery

 B) Transgenic = organism with foreign DNA  1) DNA microinjection into egg  2) all cells of organism have new DNA