DNA sequencing & DNA manipulation techniques

Restriction enzymes

-Proteins that act like molecular scissors & cut the sugar backbone of the DNA in a specific region → restriction site

-Occurs naturally in some bacteria

Once restriction enzymes cut DNA is left with…

-Blunt end fragments

-If there are no overhanging nucleotide, DNA ligase is able to join them with ANY OTHER DNA fragment that also has a blunt end

-Contains an overhang of complimentary base pairs

Sticky end fragments:

-Produced when restriction enzymes cut between the same pair of nucleotides —> results in an overhang of nucleotides

-DNA ligase can only join another fragment of DNA that also contains an overhang of complimentary base pairs

Polymerase chain reaction (PCR):

-A method used for amplifying DNA to produce large quantities in a short amount of time

What are the stages of PCR?

-Denaturation —> heating separates the DNA strands by breaking H bonds

-Annealing —> cooling allows primers to bond to the complimentary regions of the template DNA

-Extension —> DNA polymerase binds to each primer & moves along the template strand adding free nucleotides —> creates two copies of the original DNA template

Gel electrophoresis:

-Isolating & separating DNA segments/fragments based on their size & charge

What is the process of gel electrophoresis?

1. DNA fragments & dye are loaded into wells at one end of an agarose gel

2. Gel is covered in a solution containing ions, which allows the movement of DNA fragments when an electric field is applied to the gel through a negative electrode at the well end and a positive electrode at the other end

3. The negatively charged DNA migrates from the wells at the negative electrode of the gel towards the positive electrode

4. The dye samples travel faster than DNA

5. At the end, when the gel has been removed, a different dye is used that attaches to DNA can be added to reveal where the DNA fragments are

Types of genetic technologies:

-Cloning

-Recombinant DNA Technology (molecular cloning)

-Preimplantation Genetic Diagnosis (PGD)

-Stem Cells

-CRISPR (genome editing)

Gene cloning:

-Involves inserting a specific gene into bacteria —> acts as micro-factories, and produce large quantities of the protein

-Used for: insulin production for diabetic, missing clotting factor for heamophiliacs

Implications of gene cloning:

-Ethical:

*animal welfare issues

*transferring of genes from one species to another

-Social:

*reduction in cost of therapeutics made by bacteria

*providing greater access to treatments

*creating jobs

-Economic:
*makes production of therapeutics relatively cheap (and in large quantities)

-Biological:

*reduced side effects

*less deaths

*Long term changes to natural selection/evolution

Therapeutic cloning & nucleus transfer cloning:

-Both involve inserting a nucleus from a somatic cell (sex cell) into a fertilised egg cell

-The original nucleus from the fertilised egg has been removed

-Creates a totipotent stem cella

Therapeutic cloning:

-Are treated so that they will grow & divide into cells of a particular type or produce a specific type of tissue or organ

Nuclear transfer cloning:

-Cells are transplanted into a surrogate or host

-Result in identical copes of the organism that supplied the donor DNA

Recombinant DNA technology:

-Genes from one organism are added to the genome of another organism

-Uses specific enzymes to cut the DNA at a certain point so that particular DNA is removed

-This DNA is then inserted into another organism using DNA ligase to paste it together

Preimplantation Genetic Diagnosis (PGD):

-Screening technology that tests embryos for genetic defects before they are implanted

-Allows for the selection of healthy embryos

Stem cells:

-Can be used to grow organs

-Must be inserted into a host to allow the organ to develop

CRISPR (genome editing):

-An enzyme used to make cuts in DNA

-Enzyme removes the section of unwanted DNA

-The cell’s own repair system then pastes the DNA back together, making changes to the DNA