Thursday 20 October 2011

20 Notes - An Introduction to Metabolism

- Metabolism is the sum of all anabolic and catabolic processes in a cell or organism that uses energy to produce work
- Potential Energy is stored energy that is eventually converted into kinetic energy
- The First Law of Thermodynamics: energy cannot be created or destroyed, it can also be converted
- A potential energy diagram shows the changes in energy during a chemical reaction, including the amount of energy required to break molecular bonds (activation energy)
- Exergonic reactions are spontaneous (change in G is negative) and endergonic reactions are not spontaneous

- The Second Law of Thermodynamics: The entropy of the universe increases with any change that occurs
- Entropy is the measure of randomness or disorder in energy or a collection of objects
- Energy and Entropy determine whether a given chemical or physical change will occur spontaneously
- Gibbs free energy: energy that can do useful work
- The order created by anabolic processes is accompanied by even greater disorder in the universe that surrounds them

- Adenonsine Triphosphate (ATP) is the primary source of energy in living cells
- Phosphorylation is the process of attaching a phosphate group to an organic molecule such as ADP (the product through hydrolysis of the terminal phosphate of ATP) which allows for the molecule to become more reactive
- Redox reaction is the coupled reaction of oxidation (losing electrons) and reduction (gaining electrons, the oxidizing agent)
- When electrons transfer through a series of increasingly stronger electron carriers (called a coupled redox equation), more free energy is released in each step
- Partial transfer of electrons is when electrons of a covalent bond move closer to a more electronegative atom (shifts in electron positions)

- Enzymes (protein catalysts) will be effective the reactant molecules are facing each other in the right orientation, and collide with enough force to break molecular bonds
- Enzymes cannot change an endergonic reaction into a exergonic reaction, it can only decrease the activation energy required
- Substrate: the reactant that an enzyme acts on when it catalyzes a chemical reaction, it binds to a specific site on the enzyme
- Competitive (try and take over the active site) and Noncompetitive inhibitors (changes the shape of the enzyme so it does not fit into the active site) both prevent enzyme activity; it can either be a poison or a enzyme activity regulator
- Allosteric regulation involves allosteric sites that bind substances that inhibit or stimulate enzyme activity and alleosteric inhibitors which maintain inactive forms of enzymes

Monday 17 October 2011

10 Things You Need to Know About BIOTECHNOLOGY!

10. Vector Cloning (5 steps)
1. Isolate the desired genes in the plasma and human DNA
2. Insert human DNA into plasmid
3. Plasmid is placed into bacteria through transformation
4. Bacteria replicates
5. Identify the clone that carries the DNA

9. Sticky Ends & Blunt Ends
- Sticky ends are in a staggered formation, and will allow the formation of hydrogen bonds with other DNA molecules that were cut with the same enzyme
- Blunt ends will not form this hydrogen bond


DO NOT USE ENZYMES THAT CREATE BLUNT ENDS (the ends will not be able to stick together)
- Ligase is used to create phosphodiester bonds that secure the DNA fusion


8. PCR (Polymerase Chain Reaction)
- Used when DNA is contaminated or very limited
- Extremely fast
- Does not require the use of any other cells
- Three Step Cycle: Heating, Cooling, and Replication
- THe DNA is incubated in a test tube with a solution of Taq Polymerase, Nucleotides, and Primers
- Heating the DNA segment induces Denaturation, which separates them into single strands of nucleotides
- Taq Polymerase elongates the strand creating its complementary strand
- Cooling the test tube binds the single stranded DNA from heating into a double-stranded  DNA

7. RFLP (Restriction Fragment Length Polymorphism)
- Uses restriction enzymes to separate DNA molecules into shorter fragments
- The fragments are differentiated and organized through Gel Electorphoresis
- DNA sequences may differ in one or more restriction sites, thus they may produce fragments of different sizes

6. Autoradiography
- Nucleic acid hybridization will use a specific probe to identify and label bands that are related to the gene of interest
- A radioactive label is placed, identifying the target fragments
- Southern Blotting transfers the patterns on the gel to a sheet of nitrocellulose paper
- Soaking the sheet into the solution with the probe allows us to visualize the bands


5. Gel Electrophoresis
- Separates macromolecules (nucleic acids + proteins) based on rate of movement, which is determined by their size, electrical charge and other properties
- The Gel Electrophoresis moves from negative to positive
- Smaller fragments are able to travel further along the Gel, while larger fragments tend to stay behind



4. DNA Sequencing
- Uses dideoxynucleotides
- ddATP, ddCTP, ddTTP, ddGTP
- Stops elongation when mixed with a growing DNA strand
- Fragments of various lengths are made
- Gel Electrophoresis is used to determine the order of the nucleotide sequence

3. Restriction Enzymes
- Cut DNA molecules at a specific point in the sequence
- EcoRI
- SmaI
- AluI
- SalI  (blunt end)
- HindIII (blunt end)
- PstI

2. Incomplete & Complete Digestion
- Enzymes that undergo incomplete digestion are less expensive
- Complete digestion will make the restriction enzymes cut at all cut sites
- Incomplete digestion will make restriction enzymes cut at random cut sites (or none at all)

1. Transcription / Translation
- Know how to transcribe and translate
- Review exons / introns
- Know how to isolate the desired gene


Monday 10 October 2011

PCR vs. DNA Sequencing (Sanger's Method)

Similarities:
- PCR and DNA Sequencing involve breaking the entire DNA Sequence into smaller fragments

Differences:
- DNA Sequencing requires special dideoxynucleotides (ddNTPs) to create fragments of the DNA of various lengths (lack of 3'-OH)
- DNA Sequencing uses Gel Electrophoresis to read the nucleotide sequence according to the various lengths
- The purpose of DNA Sequencing is to figure out the desired sequence
- The purpose of PCR is to replicate as many fragments as possible in the shortest amount of time

PCR vs. Vector Cloning

Similarities:
- Both involve the isolation and replication of a specific gene, or short segment of DNA

Differences:
- PCR does not require the process of transformation
- Vector Cloning must utilize bacteria to replicate quickly, PCR does not require the use of any other cells
- PCR does not need expensive restriction enzymes, the desired gene is already isolated
- The purpose of Vector Cloning is to isolate a gene expression, PCR focuses on the speed and amount of replication

Sunday 2 October 2011

10 Things That Will Help YOU Ace the Test

10. How Genetics Came to Be
1928: Frederick Griffith (transformation)
1947: Erwin Chargaff (series of rules about DNA composition)
            - Chargaff's rules
Early 1950s: Watson and Crick discover double helix
                       - Rosalind Franklin (first X-ray of DNA structure)
9. DNA Structure
DNA Monomer:                                                       Nitrogen Bases:

A=T  CG
3 bonds: glycosyl, phosphodiester, hydrogen bonds











8. Replication
Initiation: Helicase, Ssbp, Gyrase
Elongation: Primase, DNA Polymerase III
Termination: DNA Polymerase I, Ligase



7. 5' TATA 3' & AAUAA

Initiation: 5' TATA 3', RNA Polymerase II, Transcription Factors
Elongation: RNA Polymerase II
Termination: AAUAA

6. Pre-mRNA to mRNA
- pre-mRNA: introns (non coding segments)
- spliceosomes: cuts introns and turns them into loops
- includes snRNPs & snRNA
- mRNA: exons (useful code)




5. DNA Translation
- Codons
- Stop codons (UAA, UAG, UGA)
- Always begins with AUG (Met protein)
- tRNA + anticodons + mRNA coding = proteins
- wobble effect (45 types of tRNA instead of expected 61 types)


This is a video that clearly describes the process of translation

4. Mutations
- Point Mutations: change of one base pair of a gene (missense & nonsense)
- Insertions / Deletions: additional or missing nucleotides
- Frameshift Mutation: Leads to the rest of the DNA being messed up
- Mutagens: External causes of mutations (UV light, X-rays, etc).

3. Prokaryotes vs. Eukaryotes 
general differences in their structure and processes

2. Purines and Pyramidines
- Adenine = Thymine
- Cytosine ≡ Guanine
Purine: Adenine + Guanine
Pyramidine: Cytosine + Thymine

1.
5' -> 3'
5' -> 3'
5' -> 3'
5' -> 3'
5' -> 3'
5' -> 3'
5' -> 3'
5' -> 3'
5' -> 3'
5' -> 3'
Get the point? 

Deaf By Design

Choice. Your life is determined by choices. Choices you make, and choices other people make for you.
If you could choose between being deaf, or being able to hear, what would you choose?

Deaf By Design is an article that describes a controversial use of prenatal genetic testing. More than often, genetic diagnoses are used to predict whether a fetus will have a disability. However, some people in the deaf community are using this test to see whether their child will be deaf.

Before leaping to any conclusions, I bet most people will be thinking,

"Why on earth would anyone want to be deaf???"

The same reason you would want to hear.

From my personal perspective, I would want to hear. I can't imagine my life without music or speaking, it is a part of my culture. The majority of people have become accustomed to life with sound because they were brought up to rely on sound for communication.
But you cannot deter deaf families from wanting the same thing we do. From their perspective, they have grown up without sound. Their culture and community rely on sign language for communication. To them sign language is as effective as speaking is to us.

We watched the documentary, Sound and Fury, to see the contrasting viewpoints of both deaf and hearing communities.

Most hearing parents would fear having to raise a deaf child, because they do not know the deaf community and how to raise the child to the best of their abilities. You could say the same for a deaf family. They would fear having to raise a hearing child, because the hearing community is foreign to them.

Either way, I believe that being afraid of how to raise a child is not a reason to disregard what their child wants. Cochlear Implants are implanted onto deaf children by the discretion of their parents. My personal belief is that parents should wait until children reach a certain age where they are able to communicate their desires. If a deaf child shows with full confidence that they want a Cochlear Implant, regardless of their age, you should allow them to do what they want. Parents should be able to believe that their child is bright enough to adapt to a new environment, and also believe in their own abilities to guide them.

In the end, it's still about choices. Every person has a different system of beliefs and is entitled to make their own choices. The happiness for one person may be despair to another. All we can do is respect each other's opinions and try to understand from their point of view.