DNA Technology

1. DNA Technology

2. Biotechnology • The use or alteration of cells or biological molecules for specific applications • Transgenics • Transgenic • “changed genes” • Recombinant DNA • DNA from different species mixed together • Natural or man-made • Whole organisms or cells • Possible because the genetic code is universal • All life uses the same genetic code (A,T,G,C)

3. Amplifying DNA • Need many copies for various DNA tests from a small initial sample • Two techniques • Polymerase chain reaction (PCR) • Recombinant DNA technology

4. PCR • Done on molecules • Based on DNA replication • Rapidly replicates a selected sequence of DNA in a test tube • Used to: • Establish blood relationships • Identify remains • Convict or exonerate suspects • Look at pathogens • Identify genes • Very sensitive but easily contaminated

5. T A T A T T T A A A C G C G C G C G G C G C G C G C G C C A T A T T T A A A A T A T T T A A Nucleotides Parental molecule of DNA Both parental strands serve as templates Two identical daughter molecules of DNA Review of DNA Replication

6. Requirements for PCR • Know parts of the DNA sequence to be amplified • 2 DNA Primers • Short, lab-made single-stranded DNA • One complementary for each strand in the DNA segment to be amplified • Lots of nucleotides • Taq DNA polymerase • From Thermus aquaticus (lives in hotsprings) • A thermal cycler

7. PCR

8. Recombinant DNA (cloning) • Works in cells • Adds genes from one type of organism to the genome of another • Requires: • Restriction enzyme • Vector • Donor DNA • Host bacteria

9. Restriction Enzymes Figure 19.3a

10. Cloning Vectors • Carries DNA from the cells of one species into cells of another • Any piece of DNA into which another can be inserted

11. Types of Vectors • Which vector used depends on size of the gene to be inserted • Plasmid • One type of cloning vector • Small circle of double-stranded DNA • Occurs naturally in some bacteria and yeasts

12. Creating Recombinant DNA Molecules Figure 19.3a

13. Figure 19.3b

14. Recombinant DNA Figure 19.5

15. Selecting Recombinant DNA Molecules • 3 types of cells • Cells that lack plasmids • Cells that contain plasmids that do not contain a foreign gene • Cells that contain plasmids that have the foreign gene –WANT THESE • Plasmids can contain a gene for an enzyme that catalyzes a reaction that makes a blue color • If a foreign gene inserts in the middle of this “blue” gene, the bacteria containing the plasma will not produce the blue color • Plasmids can contain a gene for antibiotic resistance • When the antibiotic is applied to the bacterial cells, only those with the plasmid will survive

16. Applications of Recombinant DNA • Drugs (e.g. insulin) • Pure, human versions • e.g. “humulin” • Transgenic plants • a.k.a. genetically modified (GM) plants • Transgenic animals • e.g. 3xTg-AD mouse

17. Transgenic Plants

18. Transgenic Animals

19. Isolating a Gene • Make a DNA probe • Strand of DNA complementary to the one of interest • Attached to a radioactive or fluorescent molecule

20. Complementary (cDNA) Library • Represents only protein-encoding genes • Made from mRNAs • Represents the proteins being made in a particular cell

21. Making a cDNA library • Extract mRNAs from cells • Use reverse transcriptase to make DNA from RNA • Use DNA polymerase to make double-stranded DNA