Bio Note

• energy: ability to do work
• metabolism: the sum of all anabolic and catabolic processes in a cell or organism
• kinetic energy: energy possessed by moving objects
• potential energy: energy stored by virtue of an object's position within an attractive or repulsive force field
• work: the transfer of energy form one body or place to another
• the first law of thermodynamics: the total amount of energy in the universe is constant. Energy cannot be created or destroyed bout only converted from one from into another. If an object or process gains an amount of energy, it does so at the expense of a loss in energy somewhere else in the universe
• bond energy: the minimum energy required to break one mole of bonds between two species of atoms; a measure of the stability of  a chemical bond
• activation energy: the difference between the potential energy level of the transition state and the potential energy of reacting molecules
• transition state: in a chemical reaction, a temporary condition in which the bonds within reactants are breaking and the bonds between products are forming
• entropy(S): a measure of the randomness or disorder in a collection of objects or energy
• free energy: energy that can do useful work
• second law of thermodynamics: the entropy of the universe increases with any change that occurs.
• exergonic reaction: a chemical reaction in which the energy of the products is less than the energy of reactants
• endergonic reaction:  a chemical reaction in which the energy of the products is more than the energy of the reactants
• oxidation: a chemical reaction in which an atom loses one more electrons
• reduction: gain electron
• redox reation: oxidation and reduction
• reducing agent: the substance that loses an electron/ substance that causes the reduced atom to become reduced
• oxidizing agent: the substance that gains an electron/ substance that causes the oxidized atom to become oxidized
• substrate:  the reactant that an enzyme acts on when it catalyzes a chemical reaction
• active site: the location where the substrate binds to an enzyme

Grade 12 Biotech in a Nutshell

10. Restriction Endonucleases/ restriction enzymes

• act as scissors and cut specific base-pair sequence known as recognition site
• these enzymes are used to cut double stranded DNA in a predictable and precise manner
• most recognition sites are characterized by a complementary palindromic sequence.
• palindromic = bot strands have same base sequence when read in 5' to 3'
• enzymes bind, disrupts (hydrolysis reaction) the phosphodiester bond btwn two base pairs
• then hydrogen bonds in between the cuts are broken

9. Sticky/ Blunt Ends

• sticky ends= ends of DNA fragment with short s.s. overhangs, resulting from cleavage by restriction enzymes
• blunt ends= fully base paired ends of DNA fragment resulting from cleavage by restriction enzymes
• sticky ends more useful b/c it can be joined more easily to other sticky end that has been produced by the same restriction endonuclease through complementary base pairing

8. Methylases

• enzymes that add methyl group to one of the nucleotides found in a restriction endonuclease recognition site
• prevent the bacteria's immune system to cleave its own DNA

7. DNA Ligase

• glues two fragments of DNA, generated using the same restriction enzyme, together
• two fragments are naturally attracted to each other b/c of their complementary base pairs
• hydrogen bonds will form btwn the nucleotides but it is not a stable arrangement
• phosphodiester linkage must be reformed 
• DNA ligase uses condensation reaction and drives out a molecule of water to form phosphodiester bond

6. Gel Electrophoresis

• separation of charged molecules by size in a gel
• DNA -> negatively charged, relatively similar mass nucleotides
• the fragments are put in a gel where one side is +'vely charged and other -'vely
• DNA fragments are attracted to +'ve side and moves towards it
• long fragments don't go that far and small fragments move closer to +'ve side b/c they are small and can navigate through pores
• agarose = polysaccharide found in seaweed used to form gell meshwork

5. Vector Cloning/Transformation

• the fragments of DNA with sticky ends are put into a plasmid of a bacteria
• plasmids are engineered to have multiple-cloning site, a region in plasmid that contain recognition sites of a number of restriction endonucleases
• introduction of foreign DNA, usually by plasmid or virus, into a bacterial cell
• plasmids are vectors
• bacterium that readily take up foreighn DNA is called competent cell
• selective plating isolates the cells with recombinant DNA
• cloned vectors have antibiotic-resistance gene thus if transformation is successful the bacteria will be able to grow on media that contain the antibiotic

4. PCR: Polymerase Chain Reaction

• similar to DNA replication in nucleus but instead of helicase and gyrase, heat is used to separate the strands
• at 94C - 96C the hydrogen bonds are broken
• DNA primers are synthesized in lab and is used to start the elongation
• DNA primers has to be complementary to the target area to be copied
• the temperature is brought down to 50C - 65C to let the primers anneal
• Taq polymerase starts building the complementary starnds at 72C

3. RFLP: Restriction Fragment Length Polymorphism

• polymorphism= difference in DNA sequence,, coding or non-coding, that can be detected btwn individuals
• polymorphism in coding region can be identified with specific mutations. ex. sickle  cell
• RFLP= method used to compare differences in DNA fragments btwn individuals using restriction endonucleases

2. DNA Sequencing

• Sanger dideoxy method is most popular
• similar to DNA replication
• need 4 identical single-stranded DNA with radioactively labelled primer in 4 different test tubes
• DNA polymerase and a supply of free nucleotides in form of all four deoxynucleoside triphosphates (dATP, dTTP, dGTP, and dCTP) are added
• each test tube contains dideoxy analogue of one of the deoxynucleoside triphosphates (dNTPs)
• dideoxy analogue stops elongation b/c it does not have -OH group on the 3' carbon to create a phosphodiester bond
• thus, different lengths of complementary DNA is built
• the fragments can be read in a gel

1. Application

• restriction enzymes are expensive thus cannot use many in real life
• complete digestion is also unrealistic 

PCR vs DNA Sequencing

1. DNA sequencing  require DNA polymerase, primers, dNTPs (deoxynucleoside triphosphates).
PCR require Taq polymerase, primers, supply of free nucleotides.

2. DNA sequencing require radioactively labelled dideoxy analogue of one of the DNTPs to be placed in each tube. Dideoxy analogue stops replication.

In PCR, the replication stops by itself because the polymerase reaches an end.

3. DNA Sequencing is mostly used for human genome project or sequencing a specie. 

PCR is mostly used for amplifying genes for example in forensic science. 

4. PCR can be operated in one container. Everything duplicates in one test tube for example.

DNA sequencing requires 4 separate test tube because each dideoxy analogue must be put in separate compartment. 

5. PCR starts with double stranded DNA sequence then it is heated to separate and start replication.

DNA sequence starts with a primed single-stranded DNA.

PCR vs Vector Cloning

1. Vector Cloning requires a formation of recombinant DNA.

• recombinant DNA is a fragment of DNA composed of sequences originating from at least two different sources. 
• in order to carry out mass production of target gene, the foreign genes are inserted in to plasmids

PCR does not require recombinant DNA, the gene can be duplicated in a test tube without the use of medium 

2. Vector Cloning require restriction enzymes to cut the targeted genes in order to produce recombinant DNA

• enzymes recognize and cut the DNA molecule at a specific location with specific DNA sequence called restriction site

PCR require special DNA polymerase called Taq polymerase 

• Taq polymerase is isolated from bacteria living in hot spring
• they have adapted to withstand the heat thus they have the ability to function in extreme conditions

3. PCR require a three step cycle of heating, cooling and replication

• the gene is heated at a right temperature and hydrogen bonds between the two strands are broken
• then it is cooled to anneal DNA primers (synthesized in labs) 
• after, the Taq polymerase can build complementary strands

For Vector Cloning ligase is needed to seal the strand of gene and plasmid by the formation of phosphodiester bonds. From then as bacteria multiply, so is the gene. 

4. PCR can amplify DNA from a limited amount of sources. Also it is very rapid. 

Vector cloning is very fast because the bacteria divides fast/reproduce. However, it would not be efficient when it comes to the amount of sample and it isn't as fast as PCR

5. Vector cloning is easy because the scientists know a great deal about bacteria. They have great knowledge of bacteria, the sequence of plasmid, the restriction sites and the glue making it very easy to harvest plasmid and manipulate it. Also, bacteria has stale plasmid.

PCR is all very controlled by human. The special polymerase, a supply of nucleotides and short pieces of single-stranded DNA as a primers are all provided. 

Grade 12 Genetics in a Nutshell

History


10. In 1952, Hershey and Chase discovered that DNA is the hereditary information not protein.

• conducted an experiment with bacteriophage: phage tagged with radioisotopes, 32P in DNA and 35S in protein

9. In 1953, Watson and Crick discovered the double helical structure of DNA.

• with the help from the x-ray image of DNA retrieved by Franklin, they were able to figure out the width of the DNA, 2nm, and the arrangement of nitrogenous bases
• purines can only bind with pyrimidines and this explained the Chargaff's rules

DNA Structure

8. DNA consists of many nucleotides which are made up of phosphate, sugar, and base.

• nitrogenous bases: Adenine=Thymine, Guanine(triple bond)Cytosine
• sugar + nitrogenous base => Glycosyl bond (intra), sugar + phosphate => phosphodiester (intra), nucleotides + nucleotides => hydrogen bond (intermolecular)

DNA Replication

7. DNA uses semiconservative method to replicate. One old strand and one newly made strand.

6. Enzymes and proteins carry out the job of replication. => Helicase, Single-Strand Binding Protein, gyrase, primase, DNA polymerase III, DNA polymerase I, ligase

• 5' to 3' elongation!!!
• leading strand / template can code for a continuous complimentary strand, lagging strand has Okazaki Fragments 

video of the process: http://www.youtube.com/watch?v=teV62zrm2P0

Transcription

5. Messenger RNA is transcribed from the template strand of a gene.

• transcription factors aka TFs (proteins) bind to the promotor region (TATA box)
• then RNA polymerase binds to TFs creating transcription initiation complex
• RNA polymerase II conduct elongation 
• transcription process stops after transcribing the terminator sequence, AAUAA

Video: http://www.youtube.com/watch?v=SMtWvDbfHLo&feature=relmfu

4. pre-mRNA is modified after transcription in eukaryotic cells

• 5' => 5' cap of guanine. protect form hydrolytic enzymes and signal for ribosomes
• 3' => poly(A) tail. same as 5' cap and also export mRNA from the nucleus
• RNA splicing => cut introns and join extrons to form mRNA molecule with only coding sequence
• snRNA + proteins = snRNPs + other proteins + pre-mRNA => spliceosome 

Translation

3. initiation: mRNA, tRNA with amino acid, two ribosomal subunits join together

2. Elongation

• codon recognition: elongation factor => hydrogen bonding between mRNA codon under A site with anticodon of tRNA
• peptide bond forms with polypeptide in P site and the amino acid in the A site
• translocation: tRNA with polypeptide is moved from A site to P site by ribosome and the tRNA that was previously in P site is now in E site and then leaves the ribosome

1. Termination

• release factor enters A site then hydrolyzes the bond between polypeptide and its tRNA then translation complex disassembles

Deaf By Design

Few days ago in my grade 12 bio class, we had a heated discussion about an article called "Deaf by Design".The issue evolved around a minority of people within a group of people with mutations in genes responsible for proteins called connexin 26 and connexin 30, the deaf people. In the Deaf community there are some who wants to have deaf babies just like themselves so they do genetic diagnosis and prenatal testings to make sure that they have a baby who inherited both copies of the mutated gene. So the question becomes 'is it right to let the deaf people have "disabled" babies on purpose?'

So I have created a summary of two opposing sides of the story... 

From the perspective of hearing people:

• the children did not have a choice in the decision, it is not fair and immoral for the children to have disabilities just because the parents wanted their children to be same as them
• for deaf people, it is harder to live in a world mostly full of hearing people and is mostly designed for hearing people
• by being deaf, it limits one's potential to get a job, to get a spouse, to communicate with people outside of the Deaf community or to merely have a normal life

Overall, hearing people are not hundred percent content that there are some people in Deaf community that are willing to let their children walk on more difficult road in life.

From the perspective of deaf people:

• even with the lack of hearing, deaf people live happy lives
• there is a tight community who supports each other like family and is more connected 
• there is no correct definition of "normal". Just because people can't hear does not mean they are not "normal" or cannot live "normal" lives
• deaf people does not necessarily feel "disabled". To eyes of people who has been hearing for their entire lives, it may seem like a tougher life but people who are born deaf have no trouble living as a deaf person
• it is a discrimination against "disabled" people if the society allows to abort "disabled" babies but not "normal" babies
• if deaf couple has a hearing baby, it is so easy to feel disconnected with their child because the child can easily block out the parents

Overall, deaf people feels as if there is nothing wrong with being deaf, in fact, it will create more intimate bond between the child.

It may be true, to some degree, that deaf people will have harder time in life. However, it is not because they have a mutated gene, it is because the society that we live in is more acceptable to hearing people, the "normal" people.