Introduction to DNA Cloning

1. Introduction to DNA Cloning Nucleotides and DNA Structure

2. Learning Objectives • Understand the double helix structure and dimensions of DNA molecule. • Understand the chemical bonds of the DNA molecule (which are covalent and which are hydrogen). • Understand complementary base-pairing rules of the DNA molecule and be able to predict the opposite strand. • Understand the antiparallel nature of DNA molecule.

3. DNA History • Deoxyribonucleic acid, or DNA, was discovered in the late 1860s. • It was ignored because it seemed too simple: A,C, G, T. • That’s because they degraded it when they purified it. • In the 1940s scientists discovered that chromosomes, which carry hereditary information, consist of DNA and proteins. • Experiments conducted throughout the 1940s showed that DNA actually seemed to be the genetic material.

5. Happy 51th Birthday DNA Structure

6. The first X-ray photograph of crystalline DNA in the A form. Taken by Rosalind Franklin, 1952

7. Evidence for a Double Helix • Rosalind Franklin, working with Maurice H.F. Wilkins, studied isolated fibers of DNA by using the X-ray diffraction technique, a procedure in which a beam of parallel X rays is directed on a regular, repeating array of atoms. • Watson saw pictures when Wilkins showed them at a talk, without Franklin’s knowledge.

8. Franklin’s Data • The diffraction patterns obtained by directing X-rays along the length of drawn-out fibers of DNA indicated that the molecule is organized in a highly ordered, helical structure. • The data showed DNA was a helical structure which had two distinctive regularities of 0.34 nm and 3.4 nm along the axis of the molecule. • It looked like a double helix.

9. Watson and Crick • Linus Pauling built a model of an alpha helix for protein structure and won the Nobel Prize. • Watson and Crick were inspired that Pauling used his imagination and molecular models to deduce this protein structure. • They believed if an eminent scientist such as Pauling could model a structure with little experimentation, then they might be able to do the same with DNA.

10. What Watson and Crick Knew Main components of DNA: • Phosphates • Sugars • Four nitrogenous bases • Adenine • Thymine • Cytosine • Guanine • Using wire and pieces of metal, Crick and Watson began building scale models of DNA.

11. Chargaff’s Rules • Chargaff had found that the amounts of adenine and thymine were approximately equal and the amounts of guanine and cytosine were also approximately equal. • This information gave Watson and Crick the idea that the bases might be paired in a specific way.

12. Base Composition Can Differbut Chargaff’s Rules Still Hold.

13. Failure at First • At first, Watson imagined that the bases paired like with like, for example adenine with adenine, and cytosine with cytosine. • The resulting model did not fit Franklin’s X-ray data. • Then Watson and Crick discovered that thymine and guanine could adopt a slightly different chemical shape, and their models used the wrong version of the bases.

14. Success • Using the new forms, Watson discovered that he could make two base pairs, one consisting of adenine and thymine, and the other consisting of guanine and cytosine, that had exactly the same size. • They built the model and wrote the paper.

15. Watson and Crick discovered the structure (or solved it) without direct experimentation themselves. • They read, thought and talked their way to a Nobel Prize.

16. The Solution • The DNA molecule consists of two polynucleotide chains wound around each other in a right-handed double helix. • Viewed on end, the two strands wind around each other in a clockwise (right-handed) fashion.

18. 2. The diameter of the helix is 2 nm or 20 Å 2 nm

19. The Solution (con’t) 3. The two chains are antiparallel (= show opposite polarity). • The two strands are oriented in opposite directions with one strand oriented in the 5' to 3' way, while the other strand is oriented 3' to 5'.

20. Three components of each nucleotideFour different nucleotides in DNA

21. Chemists name carbon atoms within the ring structures of organic molecules: C1, C2, C3 etc. When there are two rings in a structure, they name one ring “prime. In nucleotides, the base is one ring (C1, C2 C3 etc) and the sugar was named prime. The 5’ carbon has the phosphate and the 3’ carbon has an OH group on the carbon.

23. The Solution (con’t) 4. The sugar-phosphate backbones are on the outsides of the double helix, while the bases are oriented toward the central axis. • The bases of both chains are flat structures oriented perpendicularly to the long axis of the DNA; that is, the bases are stacked like pennies on top of one another (except for the "twist" of the helix).

26. The Solution (con’t) 5. The bases of the opposite strands are bonded together by relatively weak hydrogen bonds. • The only specific pairings are A with T (two hydrogen bonds) and G with C (three hydrogen bonds). • The A-T and G-C base pairs are the only ones that can fit the physical dimensions of the helical model. • The specific A-T and G-C pairs are called complementary base pairs, so the nucleotide sequence in one strand dictates the nucleotide sequence of the other.

28. The Solution (con’t) 6.The base pairs are 0.34 nm apart in the DNA helix. • A complete (360 degrees) turn of the helix takes 3.4 nm; therefore, there are 10 base pairs per turn. • Each base pair, then, is twisted 36 degrees clockwise with respect to the previous pair.

29. 0.34 nm 2 bp distance 3.4 nm, 10 base pairs per turn

30. The Solution 7. Because of the way the bases bond with each other, the two sugar-phosphate backbones of the double helix are not equally spaced along the helical axis. This results in grooves of unequal size between the backbones called the major groove (the wider groove of the two) and the minor groove (the narrower groove of the two). Both of these grooves are large enough to allow protein molecules to make contact with the bases.

32. DNA and RNA as Chemicals • Chemical Bonds • Covalent (permanent) • Ionic (salt) • Hydrogen (sharing H, very weak) • Van der Waals Bonds • Hydrophobic Interactions

33. Charge in Molecules • Water is perhaps the most obvious example of a molecule with partial charges. The symbols delta+ and delta- are used to indicate partial charges.

34. Covalent Bonds • Covalent Bonds are the strongest chemical bonds, and are formed by the sharing of a pair of electrons. • Once formed, covalent bonds rarely break spontaneously. • Covalent bonds don’t fall apart when heated or dissolved in a solvent like water.

35. Ionic Bonds • Ionic bonds are formed when there is a complete transfer of electrons from one atom to another, resulting in two ions, one positively charged and the other negatively charged.

36. Hydrogen Bonds • Hydrogen bonds are formed when a hydrogen atom is shared between two molecules.

37. Double strands of DNA are held together by hydrogen bonds. • The DNA molecule is usually double-stranded, with the sugar-phosphate backbone on the outside of the helix. • In the interior are pairs of nucleotide bases, holding the two strands together by hydrogen bonds. • Hydrogen bonding between the bases is specific. The adenine base can pair only with the thymine base, and the guanine base can only pair with the cytosine base.

39. Van der Walls Interactions • Van der Walls interactions are very weak bonds formed between nonpolar molecules or non-polar parts of a molecule. The weak bond is created because a C-H bond can have a transient dipole and induce a transient dipole in another C-H bond. H H | ~~~~ | CH3 CH3

40. Hydrophobic Interactions • Nonpolar molecules cannot form H-bonds with H2O, and are therefore insoluble in H2O. • These molecules are known as hydrophobic (water hating), as opposed to water loving hydrophilic molecules which can form H-bonds with H2O.

41. If thymine makes up 15 percent of the bases in a certain DNA sample, what percentage of bases must be cytosine?

42. If thymine makes up 15 percent of the bases in a certain DNA sample, what percentage of bases must be cytosine? thymine = 15%, then adenine = 15% A + T = 30%, then G + C = 70% So, cytosine is 1/2 of 70% = 35%

43. A certain segment of DNA has the following nucleotide sequence in one strand:5’ ATTGGCTCT 3’What must be the sequence of the other strand (label its 5’ and 3’ ends)?

44. A certain segment of DNA has the following nucleotide sequence in one strand:5’ ATTGGCTCT 3’What must be the sequence of the other strand (label its 5’ and 3’ ends)? Writing in the same direction: 3’ TAACCGAGA 5’ Writing 5’ to 3’: 5’ AGAGCCAAT 3’

45. How many bases are there in 2 kb (2000 base pairs) of DNA?

46. How many bases are there in 2 kb (2000 base pairs) of DNA? 4000 bases in all.

47. For the DNA strand 5'-TACGATCATAT-3' the correct complementary DNA strand is: A 3'-TACGATCATAT-5' B 3'-ATGCTAGTATA-5' C 3'-AUGCUAGUAUA-5’ D 3'-GCATATACGCG-5’ E 3'-TATACTAGCAT-5'

48. Correct Answer is: B 3'-ATGCTAGTATA-5' 5’-TACGATCATAT3’ 3'-ATGCTAGTATA5’ This choice has the correct polarity and complementarity.

49. DNA Structure as a Ladder The curving sides of the ladder of DNA represent the sugar phosphate backbone. The rungs are the base pairs. The spacing between the base pair rungs is 3.4 Å (Angstroms are 1-10 or 1/10,000,000,000 of a meter or 1/10 nanometer). The helix repeat distance is 34 Å, meaning there are 10 base pairs per turn of the helix. The strands are antiparallel. If one has 5’ to 3’ polarity from top to bottom, the other must have 3’ to 5’ polarity from top to bottom. The helix is 20 Å across at the base.