DNA Technology Ch 13

Restriction Enzymes Cut DNA ONLY at specific sites Sites identified by nucleotide sequence Restriction enzymes found in Bacteria Bacteria use them as immune system to cut up and destroy virus DNA

Vectors Things use to transport genes into cells

Plasmid Vectors A. Plasmid :small circle of bacteria DNA 1. contain only a few genes 2. bacteria take in plasmids they find in environment 3. only bacteria take in plasmids

Virus Vectors Viruses inject their DNA into nucleus of host cell We can replace virus DNA with genes we want placed in a cell Used in gene therapy 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)

Probes Radioactive tags to locate certain genes on DNA

DNA Isolation – = separate the gene you want from the rest of the DNA 1) Use probes to label DNA Cut DNA using restriction enzymes Separate DNA using electrophoresis Use only DNA that includes the radio-labeled probe Reduces volume of unwanted DNA

PCR = polymerase chain reaction Makes many copies of DNA

https://www.youtube.com/watch?v=HMC7c2T8fVk&feature=fvwrel https://www.youtube.com/watch?v=Kuy4PDb6bdU

Electrophoresis separates DNA based on size of fragment Multiple samples of DNA DNA cut with same restriction enzymes Dye added to visualize DNA Samples placed in wells of gel (indentations) Gel placed in liquid buffer

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

Gel analysis DNA standards of known length are in lane 1 Measure distance each segment moved Graph distance vs size Best fit line Estimate unknown size

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

DNA Fingerprinting steps 1. Cut with same enzyme 2. PCR to amplify DNA 3. electrophoresis :sort DNA by size 4. Compare results to crime scene or potential parents Note ……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 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 Insert foreign genes into other individual or other species Modify genes and put back in Most simple : Bacteria take in plasmids Most complex :transgenic organisms – zygote modified

Bacterial Transformation bacteria take in new genes on plasmids Happens in nature in genetic engineering using vector plasmids ( pre established plasmids used)

Vector plasmids

Creating recombinant plasmids Isolate and amplify gene of interest Cut vector plasmid and GoI with same restriction enzyme Fuse GoI and plasmid with DNA ligase to make Hybrid DNA plasmid

Hybrid DNA in plasmid vector

Creating competent bacteria Competent bacteria are able to take in plasmids. Some bacteria are naturally competent Others can be made competent by treating with cation solutions, temperature extremes or electricity

Gene Expression Competent bacteria take in plasmid vectors Hybrid genes on vector plasmid are transcribed and foreign proteins are produced