Energy

• In a local closed system, orderliness can arise without entropy increasing. However, the energy used to maintain this order will be transferred to an unusable form, hence causing entropy to increase outside the system. 
• In chemical reactions, entropy increases when
• Solids become liquid/gas products (or liquid becomes gas products)
• x moles of reactant molecules form more than x moles of product molecules
• Complex molecules form simpler molecules
• Solutes diffuse to achieve a homogeneous mixture 

Metabolism

• Energy in a reaction

• 1) Activation energy is absorbed by the reactants to break bonds 2) Reactants enter transition state 3) As products form by bonding, energy is released
• Product is more stable (bond energy is higher) than reactants 
• Change in Heat energy is negative = Exothermic reaction; Change in heat energy is positive = Endothermic
• Free Energy (usable energy) in a reaction
• Change in Gibbs free energy is negative = Exergonic reaction [spontaneous]; Change in Gibbs free energy is positive = Endergonic reaction [not spontaneous]
• Metabolic Reactions
• ATP + ATPase --> ADP + Pi + Energy
• Energy is usually not released as heat; Instead, it is coupled with an endergonic reaction 
• Redox reaction
• Involves oxidation and reduction
• A series of redox reactants result in the final oxidizing agent (that gets reduced) being the strongest 

Enzymes

• Reduces activation energy required for a reaction
• Enzymes are inhibited by competitive inhibitors (resembling the substrate) and noncompetitive inhibitors (alters binding site by binding to another site)
• Allosteric sites
• Activators: bind to these sites to stabilize an active form of the enzyme
• Inhibitors: bind to these sites to stabilize an inactive form of the enzyme
• Feedback Inhibition: Substrate --enzyme 1--> Intermediate A --enzyme 2--> Intermediate B --enzyme 3 --> end product...............end product inhibits enzyme 1.
• If end product concentration decreases, less enzyme 1 is inhibited and more end product is produced. 

Gel Electrophoresis

• Used in vector cloning (to isolate the gene of interest), RFLP (to compare fragments), DNA Sequencing

Vector Cloning (Overview, RE)

• Required materials: pBluescript vector plasmid, Restriction Enzymes, Ligase, Bacteria
• Steps
• Cut donor DNA with restriction enzymes, find the targeted DNA fragment through gel electrophoresis 
• Remove section of pBluescript using compatible RE, attach DNA fragment into plasmid at sticky ends, Bind the 2 pieces of DNA using ligase
• Reintroduce pBluescript into the bacterial vector through transformation
• Select bacteria that contains the targeted gene
• Introduce the selected bacteria to a host 

RFLP (Restricted Fragment Length Polymorphism)

•  Steps
• Cut DNA using RE (blunt)
•  Gel electrophoresis
• Southern blotting (or hybridization) onto a autoradiogram
• Compare sequences of interest 

PCR (Polymerase Chain Reaction) 

• Materials: Taq Polymerase, DNA primers, dNTPs, PCR Machine 
• Process: heat --> Cool --> Slight heat = 1 cycle
• The required piece of DNA will be obtained after the 3rd cycle 

DNA Sequencing

• Materials: DNA Polymerase, Radioactive DNA primers, dNTPs, ddNTPs
• All lanes (A T C G) will contain strands of DNA with its complete set of nucleotides 

PCR vs Vector Cloning

• Both amplify a sequence of DNA
• Vector Cloning is concerned with the protein as the end result, while PCR is only concerned with the DNA sequence. In fact, the sequence of concern in PCR might not even code for a protein.
• PCR replicates DNA through artificial methods, while Vector Cloning replicates DNA by exploiting bacteria's natural mechanisms.

PCR vs DNA Sequencing

• PCR requires a little amount of DNA, while DNA sequencing requires a lot. 
• The central idea of both techniques exploit the process of DNA replication 

When transcribing from the student's perspective (not the RNA polymerase II), do the following:

1. Find 5'TATA3'. It will be on both coding and template strands.
2. Look 5' to 3' on each strand and find AATAA. This will be the coding strand.
3. Look for intronic sequence by focusing on one strand of the intronic sequence. Look for it by running in the same direction indicated by the intron along one strand of the DNA. Then run along the other strand.

*Remember to also transcribe AAUAA on the pre-mRNA, but not to include the TATA box

In the following relationships, = indicate the same, while # indicates complementary:
Anticodon (in order of translation, ie.5' to 3' on mRNA) # 5'mRNA3' = 5'coding strand3' # 3'template strand5'
Hence Anticodon (in order of 5'mRNA3') = 3'template strand5'