Answers in Studying Molecular Biology

1. Answers in Studying Molecular Biology How does human genetic information differ from our closest relative? FOXP2 that encodes for a speech/language development protein split from the ape linage 6 million years ago

2. Answers in Studying Molecular Biology Are humans genetically programmed to contract certain diseases? Healthy Lung Cystic fibrosis with an occurrence of 1 in 3,000 for U.S. babies is caused by a defective protein resulting in high lung secretions Cystic Fibrosis Lung

3. DNA: The Genetic Code Deoxyribonucleic acid (DNA) was identified in 1944 as the information required to transform normal bacterial strains to pathogenic ones In 1951 DNA and not proteins was confirmed as the infectious agent for bacteriophages (viruses that infect bacteria)

4. DNA: The Genetic Code Chargaff’s rule (A+G = C+ T) Watson and Crick DNA structure elucidation (1953)

5. Bases that Constitute DNA At what position does the purine or pyrimidine base link with the ribose sugar backbone?

6. Nucleotides (NTPs) versus Deoxy-nucleotides (dNTPs) RNA ribose sugar DNA 2’-deoxyribose sugar What is the difference between a nucleotide and a nucleoside?

7. DNA Double Helix Stability Base π stacking and Base pair H-bonding

8. DNA Structural Features Anti-parallel polynucleotide strands Right-handed helix Sugar-phosphate exterior exposed to solvent Mg+2 stabilizes phosphate groups Two unequal grooves

9. Axial View of DNA Sugar-phosphate exterior exposed to solvent

10. DNA Melting Curve UV absorption greater with double- versus single-stranded DNA

11. DNA Re- and De-naturation DNA can re-nature with single strands re-forming a double helix if cooling occurs slowly. In nature, DNA can be unwound by helicases and cut by endo- or exo-nucleases at phosphodiester bonds

12. DNA Melting Point Depends on GC Content

13. RNA versus DNA Components Sugar hydroxylation pattern Base methylation pattern

14. RNA Structural Features Less stable Normally single stranded Can fold and base pair with complementary segments of the same strand Transfer RNA molecule

15. Other Nucleotides Molecules/Functions Acetyl unit transferase + R-C(O)CH3

16. Other Nucleotides Molecules/Functions Oxidation/Reduction Reactions

17. Other Nucleotides Molecules/Functions Oxidation/Reduction Reactions

18. Central Dogma of Molecular Biology DNA, RNA and proteins, are linear polymers (i.e. each monomer is connected to at most two other monomers) The monomer sequence effectively encodes information

19. DNA Replication Proteins catalyzed: [i] unwinding of the super helix – DNA helicases [ii] unwinding of the double helix – DNA helicases [iii] replicate the master template – DNA polymerases

20. DNA Transcription RNA polymerase and transcription factors catalyze messenger RNA (mRNA) formation from DNA sections In eukaryotes the primary transcript (pre-mRNA) is processed via alternative splicing

21. Nucleotides (NTPs) versus Deoxy-nucleotides (dNTPs) RNA ribose sugar DNA 2’-deoxyribose sugar What is the difference between a nucleotide and a nucleoside?

22. RNA to Protein Conversion: Translation Mature mRNAs attach to ribosomes where it is read as a triplet codon Initiation and elongation factors bring aminoacylated transfer RNAs (tRNAs) into the ribosome-mRNA complex

23. Standard Amino Acid Genetic Code

24. Cystic Fibrosis Protein Function/ Gene Structure Diagnostic signs of CF include: High chloride concentration in sweat Thick mucus in the airways

25. A Mutation Site Resulting in Cystic Fibrosis

26. Sanger or Dideoxy DNA Sequencing • Components for the Chain Termination Method: • DNA fragment for sequencing • Primers • dNTPs • ddNTPs

27. Sanger or Dideoxy DNA Sequencing What direction does the DNA polymerase elongate the DNA fragment?

28. Sanger or Dideoxy DNA Sequencing Migration smaller is faster through the gel (bottom smallest) Each peak represents 1 base

29. Pyrosequencing Based on DNA synthesis Template DNA is immobilized Peak area represents base number http://www.pyrosequencing.com/DynPage.aspx?id=7454

30. Gene Amplification by Polymerase Chain Reaction (PCR) Gene specific amplification without purification Amplification via temperature cycling Special Taq (Thermus aquaticus) DNA polymerase Detection of Helicobacter pylori, Borrelia burgdorferi, as well as hepatitis, HIV, and West Nile virus

31. Restriction Enzyme Recognition Sites Restrictive endonucleases recognize 4-8 bp sequences within the DNA and cleave at a specific site DNA strands that are cut in a symmetric fashion are palindromic Blunt and Sticky DNA Cuts

32. Molecular Cloning by Recombinant DNA Technology • Cut by restriction enzymes • Anneal an uncatalyzed reaction • Re-close by DNA ligase

33. Screening for Colonies Containing the Plasmid with the Insert

34. Size-Specific Cloning Vectors

35. Recombinant Protein Products

36. Generating a Restriction Map Where are the restriction sites located on the 20 kb fragment that would generate this map? . . .

37. Transcript Analysis via Microarray/DNA Chip How to examine gene expression changes with a given treatment mRNA ↓ cDNA fluorecent labeling ↓ cDNA/oligonucleotide Hybridization ↓ Slide scanning ↓ Data analysis

38. Differential Arabidopsis Gene Expression with GB03 Exposure RNA Extraction cDNA Synthesis Label Hybridize Wash Scan Block 8 Full Image

39. cDNA Synthesis and Labeling Amino Allyl-dUTP Cy5 Dye

40. Microarray DataAnalysis

41. Chapter 3 Problems: 1, 2, 7, 8, 9, 13, 15, 19, 21, 25, 29, 61, 62,