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

Are humans genetically programmed to contract certain diseases? Answers in Studying Molecular Biology 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 Healthy Lung Cystic Fibrosis Lung

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)

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

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

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

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

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

Axial View of DNA Sugar-phosphate exterior exposed to solvent

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

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

DNA Melting Point Depends on GC Content

RNA versus DNA Components Sugar hydroxylation pattern Base methylation pattern

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

Other Nucleotides Molecules/Functions Acetyl unit transferase + R-C(O)CH 3

Other Nucleotides Molecules/Functions Oxidation/Reduction Reactions

Other Nucleotides Molecules/Functions Oxidation/Reduction Reactions

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

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

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

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

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

Standard Amino Acid Genetic Code

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

A Mutation Site Resulting in Cystic Fibrosis

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

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

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

Pyrosequencing Based on DNA synthesis Template DNA is immobilized Peak area represents base number

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

Restriction Enzyme Recognition Sites Blunt and Sticky DNA Cuts 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

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

Screening for Colonies Containing the Plasmid with the Insert

Size-Specific Cloning Vectors

Recombinant Protein Products

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

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

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

cDNA Synthesis and Labeling Amino Allyl-dUTP Cy5 Dye

Microarray Data Analysis

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