1. DNA Technology
Ch 13

2. 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

3. Vectors
Things use to transport genes into cells

4. 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

6. 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)

7. Probes
Radioactive tags to locate certain genes on DNA

8. 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

9. PCR = polymerase chain reaction
Makes many copies of DNA

11. 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

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

13. 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

14. 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

15. DNA Fingerprinting Uses
Crime scene investigations
Paternity tests

16. 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

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

18. 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

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

21. 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

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

23. Vector plasmids

24. 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

25. Hybrid DNA in plasmid vector

26. 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

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