Vector Cloning

Vector Cloning:
- gene of interest is first cut out and removed from a cell
- a bacterial vector is also cut using the same enzyme to create two sticky ends
- the sticky ends is used to connect the original vector to the new gene from the original host
- newly formed vectors, recombinant DNA, (containing ampicillin resistance genes) are placed with the bacteria E. Coli, and some E. Coli cells will pick up the new vector through the process of transformation
- the bacteria cells will then be placed in a container with yeast as their nutrient and ampicillin
- bacteria cells without ampicillin resistance genes would be killed while the one that received the new vector would grow slowly
- the surviving "desired" bacteria cells, those with the recombinant DNA, are then being cloned

PCR Cloning

Polymerase Chain Reaction (PCR)
- it is the direct method of making copies of a desired DNA sequence
- it is similar to DNA replication in a way that they both occur in the nucleus
- heat (94°C ~ 96°C) is used to seperate the hydrogen bonds between complementary bases, thus creating two template strands
- temperature is then cooled off to about between (50°C ~ 65°C) to allow the primers to anneal
- DNA primers are used instead of RNA primers and attach to the 3' end of the gene of interest
- two primers (forward and reverse) are extended by using Taq polymerase after heating to 72°C
- Taq polymerase then synthesizes the complementary strands from free nucleotides
- steps are repeated for the two newly created sets of semi-conservative DNA

For clearer resolution of the iamge below click on http://www2.le.ac.uk/departments/emfpu/genetics/explained/images/PCR-process.gif

10 Things to Know About Replication

1) Protein Helicase unwinds the double helix by breaking down the hydrogen bonds between the template and coding strand.


2) Single Stranded Binding Protein stabilizes the single stranded DNA.


3) Gyrase releases the tension and pressure on the DNA as it unwinds.


4) At the same time a replication fork, a section on the DNA where the two strands are still joined, is created. When two forks are close enough to each other a bubble, known as the replication bubble, is formed.


5) Leading strand (5' to 3') and lagging strand (3' to 5') are formed. Okazaki fragments appear on the lagging strand.


6) RNA Primase signals the Polymerase III by producing Primers which are also RNA.


7) DNA Polymerase III recognizes Primers and elongate the new DNA from 5' to 3'.


8) DNA Polymerase I replaces the Primers with DNA and proof read the new DNA.


9) Ligase fill in the gaps in the DNA.


10) A new and identical DNA is formed.

Notable Geneticists

T.H. Morgan and his group of scientists discovered genes are the chromosomes as well as determined proteins and DNA as candidates for genetic material.

Frederick Griffith discovered the genetic role of DNA in 1928. He made the famous experiment of using two strains of one bacteria known as Streptococcus pneumoniae. The two strains consist of a harmless R strain and a pathogenic S Strain. A mouse would survive from a harmless R strain as well as a dead S strain. However, when the two harmless elements were fused together and injected into the mouse, the mouse died from a pathogenic S strain. The newly discovered the phenomenon, transformation, was responsible. This is the result of a change in genotype and phenotype due to the assimilation of a foreign substance (now known as DNA) by a cell.

In 1947, after studying the DNA composition in organisms, Erwin Chargaff developed a series of rules. He found out that the DNA composition varies from species to species. Furthermore, although the 4 bases (A,T,G,C) are found in characteristic in any one species, the bases do not appear in equal amounts nor ratios. Chargaff's Rules was developed to reflect his findings on the ratio of nucleotide bases. Adenine equals approximately to Thymine, while Guanine equals approximately to cytosine.

James D. Watson and Francis Crick were a student-teacher group working to solve the 3-D DNA structure. By the 1950s, numerous groups of scientists were in the race to discover the true structure of DNA. After receiving the findings of Wilkins and Franklin, Watson managed to learn that the DNA was of helical shape. Watson deduced the width of helix and the spacing of bases to produce the first successful 3-D DNA structure.

Maurice Wilkins and Rosalind Franklin used X-ray crystallography to study the structure of DNA. Unfortunately, Franklin died at an early age to to excessive radiation exposure.   

Gr 12 Biology Terms

Genetics & Structures:

Chromosomes: an organized structure of DNA and protein that is found in cells.

Chromatids: two identical copies of DNA making up a duplicated chromosome.

Chromatins: a mass of genetic material composed of DNA and proteins that condense to form chromosomes.

Homologous Chromosomes: chromosome pairs of the same length, centromere position, and staining pattern, with genes for the same characteristics at corresponding loci.

Homologs: a homologous trait, often refer to a homologous protein, and to the gene (DNA sequence) encoding it.

Replicated Chromosome: a replicated chromosome has undergone DNA replication and contains
two sister chromatids.

Unreplicated Chromosome: cell's original number of chrosome (6).

Sister Chromatids the two identical strands of a single replicated chromosome. 

Sister Chromosomes: 2 daughter cells, with its own nucleus and identical chromosomes.

Centromeres: the constricted region joining the two sister chromatids that make up an X-shaped chromosome.

Centrioles: a self-replicating, small, fibrous, cylindrical-shaped organelle, typically located in the cytoplasm near the nucleus in cells of most animals.

Centrosome: the organelle located near the nucleus in the cytoplasm that divides and migrates to opposite poles of the cell during mitosis.

Ideas for ISU

Biology ISU is just around the corner and it's time to be brainstorming some ideas regarding possible topics. As Mr. Chung insinuated, the topic of the project can sometimes be more effective than the actual effort put into it. The biggest three categories are probably: animals, bacterias/viruses, and plants. Actual plants experimentation could take a long time and it's not really my most loved life form so I will stray away from plant ideas. Animals and microorganisms are more appealing to me and they are more research based than experiments. These sub topics could be further broken down to behavioral/psychological and biological aspects.

Possible Topic:
Which is more motivating: punishment or reward?

Said topic focuses on the psychological aspect of the human nature, as to whether we respond better to punishments or rewards.