Advancements in DNA Technology: Mutation, Cloning, and Electrophoresis

1. DNA Technology G/T Biology I

2. Uses of DNA Technology • Purposely mutate genes to find out what they do (must mutate developing embryo) • Clone genes (put human genes into bacteria - makes a lot of copies of a gene to study it, sequence it, or make large amounts of a protein) • See if an embryo has a defective gene (use complementary DNA pieces to “tag” a gene) • Make vaccines (make large amounts of proteins that trigger the immune response) • Gene Therapy (putting genes into somatic or germ cells)

3. Uses of DNA Technology Involving Gel Electrophoresis • Identify recessive alleles • Diagnosis of disease • Study of relatedness of species • Crime Solving (DNA fingerprinting) • Paternity Testing • Figuring out the sequence of a gene

4. Restriction Enzymes – What are they and why do you need them? • They are enzymes that cut DNA at very specific sequences • We need them for any kind of recombinant DNA technology (genetic engineering) – splicing genes into different DNA • Ex. Putting the human insulin gene into bacteria so that we can get bacteria that multiply quickly to make buckets of human insulin for diabetics • We need them for any DNA technology involving gel electrophoresis

6. What is Electrophoresis? • Electrophoresis is the separation of molecules (pieces of DNA) in a porous matrix based on electrical charge and size • The porous matrix is a gel • If you put an electrical charge through the gel material and put the DNA at the negative pole, it will migrate to the positive pole since DNA is negatively charged • If you cut the DNA into pieces and then run it thru the gel, the bigger pieces of DNA will move slower, the smaller pieces will move faster – therefore the pieces of DNA will separate

10. How do we use Electrophoresis to Diagnose a Disease or Identify a Recessive Allele • Cut the DNA (if you have purified the gene) • Run gel (DNA runs toward the + pole) • Stain gel CCG↓CGGTAGGAAC CCACGGTAGGAAC ____ ____ ____

12. How do you use Electrophoresis for DNA Fingerprinting? • Even though human DNA is 99.9% the same, 0.1% is 6 million differences in individual nucleotides. • Through research, we have identified regions of the DNA that is the most variable and cut those areas with restriction enzymes • This will make different sized fragments based on the enzymes cutting the DNA differently • If we run them on a gel, we will see different patterns • The same person always has the same pattern because the DNA doesn’t change • Different people may have the same pattern if they have the same mutations in the areas of the DNA analyzed

13. This is an example of a real DNA fingerprint from a real case

14. How to Use Electrophoresis for Paternity Testing • Cut the DNA up with restriction enzymes and run it on a gel • Every allele the child has, must come from the mother or father • If you know who the mother is, any band that didn’t come from her, must be from the father

15. PCR – polymerase chain reaction • Make millions of copies of a single piece of DNA (like DNA replication in a test tube) • DNA can be old and in very small quantities • Can use for crime detection if only have 1 cell or a small sample • Basically, throw in DNA into a test tube with nucleotides, DNA polymerase • Heat it up to separate the DNA and cool it down for it base pair back together • The DNA polymerase is special – isolated from bacteria that live at 160 degree water

16. B. Denature 96º 50º C. Anneal primers 72º D. Polymerase binds A. Double strand DNA 50º Taq Taq

17. 72º 1 96º 2 F. Denature 3 4 Taq Taq E. Copy strands Taq Taq