Recombinant DNA Dr. R. Howells

1. Dental BiochemLecture 37 Recombinant DNADr. R. Howells

2. Restriction Endonucleases: enzymes produced in bacteria that typically recognize 4-8 bp DNA palindromic restriction sites; cuts can be staggered to yield single-strandedsticky ends or blunt to yield flush ends

4. Examples of True Palindromes Able was I ere I saw Elba. Madam in Eden, I’m Adam. As I pee, sir, I see Pisa! A man, a plan, a canal: Panama.

6. For each restriction enzyme, bacteria also produce a site-specific methylase that protects their own DNA from cleavage

7. Ligation of restriction fragments with complementary sticky ends

9. Basic components of a plasmid cloning vector that can replicate within an E. coli cell: exogenous DNA (up to 10-15 kbp) can be inserted into the polylinker region

10. Molecular Cloning

11. Construction of a cDNA Library

12. Polymerase Chain Reaction (PCR)

14. PCR to screen for cystic fibrosis CF is an autosomal recessive disease that affects the pulmonary and digestive systems. In >70% of patients, a 3 base deletion in the coding region of the CFTR gene (cystic fibrosis transmembrane conductance regulator) results in a F508 mutation that prevents normal folding of the channel, leading to destruction of the protein by the proteasome. The 3 bp deletion in the CFTR gene can be detected by PCR.

16. Assay of Variable Number of Tandem Repeats

17. DNA Fingerprinting

18. Sanger DNA sequencing: ddNTP vs dNTP

19. Sanger Dideoxy DNA Sequencing

21. Sanger DNA Sequencing

22. Deep DNA Sequencing Nobel Laureate James Watson, Ph.D., co-discover of the DNA double helix and father of the Human Genome Project, peeked at his own genome sequence and saw the potential for a better society. "I think we will have a healthier and more compassionate world 50 years from now due to the great technological advances we are celebrating here today," said the genomic pioneer, who became the first person to receive the data that encompass his DNA sequence in ceremonies at Baylor College of Medicine on May 31, 2007. A company–454 Life Sciences–did the sequencing using new technology that cut the time and cost a thousand fold compared to the international Human Genome Project completed in 2003. That "big science" project generated a generic genome drawn from the DNA of many people. It took 13 years and cost $2.7 billion–less than projected when it started in 1990. Sequencing Watson's genome took two months and cost $1 million.

23. Human Genome Highlights • Feat was first jointly announced June 26, 2000 at the White House • Human genome consists of 3.2 billion base pairs: if printed in standard type it would occupy 75,490 pages of the NY Times • Surprisingly there appear to be only 25,000 human genes • Only 300 genes have no counterpart in the mouse genome; the chimpanzee genome is predicted to be virtually identical to the human • 1-1.5% of genome encodes protein • humans are roughly 99.9% identical in sequence (SNPs occur about 1/1250 bp)

24. Amniocentesis and chorionic villus sampling as source of DNA for prenatal testing

25. Southern blotting

26. Southern blotting to detect Hb S

27. Northern blotting Analysis of RNA: -Level of expression -Size of transcript

29. Recombinant protein production: synthetic human proteins are useful therapeutic agents • Greater consistency of product quality • Higher efficiency of production • Lower immunogenicity than animal proteins • Reduced infectivity compared to tissue-derived products • Possible genetic engineering of “super proteins” with higher activity Advantages of recombinant protein production over purification from source (animals or human cadavers) include:

30. A sampling of recombinant proteins used clinically

31. Transgenic livestock animals for protein expression

32. Gene therapy of adenosine deaminase deficiency