Unit 3 AOS 1 - Chapter 4: DNA Manipulation

Polymerase enzyme

Enzyme that synthesizes a polymer from monomers. DNA and RNA polymerase synthesizes their respective nucleic acid. 5' to 3' direction. These require primers to stick attach to template strand

Ligase

An enzyme that connects two fragments of DNA by creating phosphodiester bonds

Restriction Endonuclease

Enzyme that cut the sugar-phosphate bond between nucleotides. They cut DNA at a specific base sequence or a recognition site

recognition site

a specific target sequence of DNA upon which restriction endonuclease act, often palindromes (important for creating sticky ends)

How are blunt ends made?

If the enzyme cuts the restriction site in the center as there are no overhangs for the DNA bases to bond to via hydrogen bonding

How are sticky ends formed?

If restriction endonuclease cut the DNA in any other location besides the center, the resulting ends have overhangs that are complementary to the opposite sticky end cut by the same enzyme, thus able to form hydrogen bonds.

Purpose of PCR

To amplify genes

requirements of PCR

- a DNA sample that subsequently gets denatured and amplified

- taq polymerase is required in the elongation stage

- nucleotide bases must be constantly available for the polymerase to create new strands

- primers join to the 3' end of a single stranded dna, allowing the polymerase to attach and begin extending the DNA strand.

primer

a short, single strand of nucleic acid that acts as a starting point for polymerase enzymes to attach

1st step of PCR

Denaturing, the DNA is heated up to high temperature of 95 degrees to break the hydrogen bonds present to split DNA into single strands

2nd step of PCR

Annealing. Temperature is lowered to 55 degrees to allow primers to bind to complementary sequences.

3rd step of PCR

Elongation/extension. The temp is heated back to 72 degrees Celsius. taq polymerase binds to the DNA and extends the primers to full strand.

Applications/use of PCR

Genetic testing, finding mutations and genetic disorders

Forensic analysis - profile individuals and identifying subjects through DNA fingerprinting

Parentage testing

Functional analysis of Genes

gel electrophoresis

a technique that separates DNA fragments based on their molecular size

1st step of gel electrophoresis

DNA samples are placed in wells at one end. A standard ladder of DNA fragments with known size is loaded to estimate the size of unknown DNA fragments. The plate is made up of agarose gel which contains pores to allow for the movement of DNA. This agarose gel is immersed in a buffer solution which helps carry an electric current.

2nd step of gel electrophoresis

an electrical current is passed through the gel using 2 electrodes, one positive and one negative. Negative near the well, positive other side. As DNA is negatively charged due to he phosphate backbone, it will be attracted to the positive electrode. When the current it applied, the DAN will move from wells through the gel to the positive electrode

3rd step of gel electrophoresis

smaller DNA fragments move faster through gel and travel further than lager ones. After a while, the current is switched off and the DNA will stop moving and settle into bands, separated based on size

4th step of gel eelctropheoresis

DNA is difficult to see with the naked eye so the gel is stained wiht a fluorescent dye such as ethidium bromide alloiwng it to be visible under UV light. this dye can be added in the gel before the experiment

importance of standard ladders

dna fragments of same size do not always travel the same distance due to different gel types and inconsistent experimental conditions influencing the distance moved by the DNA

factors that change the length travelled by DNA

voltage - stronger electrical force means a greater distance traveled due to stronger attraction

gel composition - gels with a greater density and agarose concentration increase the difficulty for fragments to move through

buffer concentration - greater the concentration of ions in the buffer the more electric current is conduced through the gel which causes DNA to move down the lane

time

Why transform bacteria

The fact that bacteria possess independently replicating plasmids means that humans can genetically modify bacteria to synthesis large amounts of protein in a simple process. This involves editing a plasmid to incorporate a target gene of interest. Bacteria take up these recombinant plasmids in bacteria transformation. Once this has occurred, bacteria can synthesise specific proteins.

1st step to making a recombinant plasmid

Choose a specific restriction endonuclease that cuts sticky ends with a restriction site located upstream and downstream of the required gene of interest (which does not contain any introns as plasmids do not contain introns)

2nd step of making a recombinant plasmid

A plasmid vector with the following 4 DNA sequences is chosen:

1. The same restriction endonuclease site located within a reporter gene (a gene with an easily recognisable phenotype) so we can tell if the recombinant plasmid has been taken up

2. An antibiotic resistance gene

3. Origin of replication to signal that start site for DNA replication

4. Reporter gene

3rd step of making a recombinant plasmid

The same restriction endonuclease used to cut the gene of interest cuts the plasmid, and ligase combines the target gene with the cut plasmid to create a recombinant plasmid. However not every plasmid will take up the gene of interest as most plasmids will simply legate back with themselves and are termed non-recombinant plasmids. As the reporter gene was the gene cut in the plasmid, it plays a big role in distinguishing between the recombinant and non recombinant plasmids

Transforming bacteria

Bacteria naturally take up plasmids from the environment however they first must be made competent to uptake these plasmids via heat shock or electroporation. Here either increasing the temperature of delivering an electric shock to the bacteria increases the permeability of the membrane and the likelihood of uptaking the plasmid.

Antibiotic selection

In order to distinguish between transformed and untransformed bacteria, the bacteria is cultured into an antibiotic rich plate. Since the plasmid contained the antibiotic resistance gene such as ampicillin resistance, bacteria growing in the plate indicate that these have successfully uptaken the plasmid. To further distinguishing between the non-recombinant plasmids and recombinant plasmids, we can test for the expression of the reporter gene. Non recombinant plasmids will display the phenotype for the reporter gene as this gene is continuous and not disrupted by the gene. Recombinant plasmids will not display the phenotype.

Protein production and extraction

The transformed bacteria are cultured and induced to produce the target protein. As the bacteria make lots of different proteins, the protein of interest is extracted and purified

The process of artificially producing insulin

The recombination, transformation of bacteria and antibiotic selection is the same except 2 plasmid vectors are used, one for insulin A and B. The ampicillin resistance gene and the tetracycline resistance gene (the reporter gene) are used. EcoR1 and BamH1 are the endonuclease.

- after determining the recombinant plasmids and transformed bacteria, the plasmids in these bacteria are taken out and cut up using EcoR1 to insert the LacZ gene which produces b-galactosidase. When expressed, it produces an insulin subunit attached to the b-galactosidase to form a fusion protein. This stops the smaller insulin subunit from being digested in E.coli

- bacterial transformation occurs and antibiotic selection begins. However this time it is placed into agar plates containing ampicillin and X-gal, as b-galactosidase converts X-gal changing from a colourless compound to being blue. Colonies that grew and were blue were identified as containing recombinant plasmids due.

Process of artificially producing insulin, 2

The transformed bacteria are placed into conditions to exponentially reproduced before their membranes were broken downs and the insulin subunit and b-galactosidase fusion protein were extracted. The methionine at the start of the insulin gene is broken down to seperate insulin allowing for isolation and purification.

- the 2 insulin chains are then mixed together which allowed the connection of disulphide bonds to from and crate functional human insulin