DNA

1. Central Dogma of Biology DNA RNA Protein

2. virulent avirulent avirulent virulent Frederick Griffith, 1928

3. T2 Bacteriophage A bacteriophage infects and reproduces inside a bacterial cell Bacteriophages contain only DNA and proteins. A.D. Hershey and Martha Chase, 1952

4. P S A.D. Hershey and Martha Chase, 1952

5. Oswald Avery, Colin MacLeod and Maclyn McCarty, 1944 Chemical nature of the transforming material Resistant to organic solvent and proteases -- it is not a protein Resistant to ribonuclease -- it is not an RNA molecule Sensitive to deoxyribonuclease -- it is likely a DNA molecule

6. DNA 1869, Friedrich Miescher discovered in the cell nucleus a mixture of compounds -- nuclein. Nuclein contains deoxyribonucleic acid (DNA). At the end of 19th century: Chemists learned the structure of DNA and a related compound, ribonucleic acid (RNA) Both long polymers of nucleotides.

7. A nucleotide is composed of a sugar, a phosphate and a base

8. Nitrogenous bases Nitrogenous bases Phosphoric acid Phosphoric acid The chemical nature of DNA and RNA DNA RNA Adenine (A) Cytosine [C] Guanine (G) Thymine (T) Adenine (A) Cytosine [C] Guanine (G) Uracil (U) Sugar deoxyribose Sugar ribose

11. nucleosides

12. nucleotides

13. 3’ 5’ 5’-3’ OH 3’ 5’

14. 5’ end 3’ end 5’ TCA 3’

16. How does DNA act as a genetic information carrier? Can store large amounts of information Can be replicated faithfully

17. Francis Crick agreed with Waston’s hypothesis. He also pointed out that because of certain bond angles and the proximity of the base pairs, the two helices had to run in opposite directions. The helices are antiparallel to one another.

18. Chargaff’s Rules Erwin Chargaff observed in 1950 that the content of purines was always roughly equal to the content of pyrimidines. Furthermore, the amounts of adenine and thymine were always roughly equal, as were the amounts of guanine and cytosine. A=T, C=G, but what does this mean?

19. X-ray structural information Rosaline Franklin, 1952 What does this interesting pattern mean? The DNA is a simple, regular structure with repeated units

20. Watson and Crick, in their manuscript (900 words) on DNA structure prediction published in Nature, they wrote: “It has not escaped our notice that the specific base pairing we have proposed immediately suggests a possible copying mechanism for the genetic material.” This basically serves as a guideline on how DNA can be faithfully replicated or copied into the next generation.

21. Francis Crick Maurice Wilkins Rosalind Franklin James Waston

22. Hydrogen bond

25. A B Z A Inclination of base pair from horizontal Residues per turn Form Pitch Å A 24.6 ~11 +19º B 33.2 ~10 -1.2º Z 45.6 12 -9º

26. Although G=C and A=T are true for every organism, the amounts of G+C contents vary from organism to organism

27. Two DNA strands can be separated simply by heating, a process called DNA denaturation or DNA melting. The temperature at which the DNA strands are half denatured is called the melting temperature, or Tm. Tm of a DNA is largely determined by its G/C%. The amount of DNA strand separation can be measured by the absorbance of the DNA solution at 260 nm light.

28. The process of reuniting the separated DNA strands is called annealing or renaturation Temperature -- 25ºC below Tm. DNA concentration -- the higher concentration, the better the annealing. Renaturation time -- the longer the time, the better the annealing.

29. annealing The process of annealing a DNA strand with a complementary or nearly complementary RNA strand or DNA strand from a different origin is called hybridization.

30. DNAs are of various sizes and shapes

31. How do we know which DNA region is important for a particular biological function? For example, the eye color the height of a person the sexual identity disease/cancer Identify, separate, and manipulate a specific DNA fragment

32. Molecular Cloning Inserting a piece ofDNA molecule (of interest)))into aDNA carrier (vector)to generate multiple copies in a host cell such asbacteria Purposes Separate a gene from others Amplification of modified forms of genetic materials Manipulation of DNA for further experiments Vector (DNA carrier) Plasmids Cosmids YAC Bateriophage Virus

33. Enzymes that can cut DNA Enzymes that can join DNA

34. Restriction Endonucleases CCCGGG GGGCCC Some make even cuts Restriction Endonucleases --The Molecular Scissors Host enzymes that prevent the invasion of foreign DNAs such as viral DNA, by cutting them up. These enzymes cut within the foreign DNAs, rather than chewing them away from the ends. These enzymes recognize a specific DNA sequence (4-12bp) which is twofold symmetry and cut both DNA strands Some enzymes make staggered cuts GAATTC CTTAAG

35. S -- deoxyribose P -- phosphate groups 5’ 3’ 5’ 3’

36. Sticky end Sticky end

38. 1. Origin of replication 2. Antibiotic-resistant genes 3. Multiple cloning sites Vectors -- the DNA carriers Capable of replicating in bacteria -- an origin of replication Allow the vector as well as the foreign DNA to amplify in the host cell Plasmids 2) Phages Allow the host to grow on selective media Can selectively amplify this specific vector in the host cell Allow insertion of foreign DNA

39. Vectors -- the DNA carriers Plasmid as a vector Host: E. coli Vector size: usually about 3kb. Insert size: up to 20kb. usually below 5 kb. Insert select: functional inactivation of the ability to resist an antibiotic

40. DNA ligase It is really a pain to do replica plating!

41. Multiple cloning sites -peptide or N-terminus of b-galactosiase

42. Bacteriophages as DNA carriers - Natural vectors that transduce DNA from one bacterial cell to another. - A virus for a bacterial cell - Cannot “live” or reproduce without getting inside a bacterial cell

44. A single phage can infect and clear out many bacterial cells and creat a “plaque” on a bacterial lawn A plaque contains a homogeneous population of a phage

45. Bacteria Bacteria Bacteria Host ~ 1-8 kb Up to 50kb ~ 12-20 kb Insert size Transformation Infection Infection Entry into Cells Less efficient Very efficient Very efficient Efficiency Outcome Multiply Multiply and kill Multiply Application Genomic library Genomic library Cloning Appearance of infected cells Colonies Plaques Colonies Cosmids, Phages, and plasmids as DNA carriers Plasmid Phage Cosmid

46. 12kb-20kb <12 kb, can not be packaged efficiently >20 kb, too large to fit into the phagehead

47. Phage replication Phage replication 12kb-20kb

48. Origin of replication 40kb-50kb Cosmids as the DNA carriers Colonies No plaques

49. Cosmids Cosmids Cosmids as the DNA carriers Cosmids behave both as plasmids and as phages They contains the Cos ends from the  phage DNA Allow the DNA to be packaged into  phage heads They also contain a origin of replication from the plasmid so they replicate like plasmid in bacteria Host: E. coli Vector size: usually about 5-7 kb. Insert size: up to 50kb

50. EcoR I EcoR I EcoR I EcoR I EcoR I EcoR I EcoR I Incomplete digestion Low enzyme concentration Complete digestion High enzyme concentration Partial restriction digestion