Restriction enzymes & Gel electrophoresis Flashcards
What are restriction enzymes?
Restriction enzymes recognizes and cuts at short and specific DNA sequence (recognition sites)
Can be found in bacteria
What are the uses of restriction enzymes?
Genetic engineering and Recombinant DNA
What is recombinant DNA?
Restriction enzyme cuts the DNA and remove a certain/target gene so that we can add in the gene we want into the bacterial plasmid
What is genetic engineering?
Bt gene has the ability to keep pests away
Bt gene is cut from bacterial DNA and a certain from the corn plant is removed, both processes are carried out by the same restriction enzyme
Bt gene is added into the corn plant’s DNA by DNA ligase
The corn plant has the ability to keep pests away by producing natural toxins
Why are recognition sites palindromic?
The sequence of recognition sites are the same when both strands are read from 5’ to 3’ direction.
How are sticky ends and blunt end formed?
Restriction enzymes will break the phosphodiester bonds on each strand of DNA double helix.
Resulting in sticky ends while other results in blunt ends
How are DNA fragments formed?
Restriction enzymes cuts the DNA at recognition sites to produce separate DNA fragments
Difference between cutting linear DNA and circular DNA
When linear DNA is cut, the number of fragments formed would be one more than the number of cuts made (e.g. 3 cuts made, 4 fragments produced)
When circular DNA is cut, the number of fragments formed would be the same as the number of cuts made (3 cuts made, 3 fragments formed)
How does gel electrophoresis work?
An electrical field is applied to move the negatively-charged DNA to the positive electrode through an agarose gel matrix.
Electrophoresis can be done because DNA is negatively charged and DNA fragments move at a different pace.
- current must go from negative to positive so that DNA can move
- if current goes from positive to negative, the DNA will not move
How is linear DNA separated? (during gel electrophoresis)
Linear DNA is separated by size
- smaller DNA fragments would migrate faster over a longer distance
- bigger DNA fragments would migrate slower over a shorter distance
What is the purpose of DNA ladder?
DNA ladder is a collection of DNA fragments of known lengths
Length of DNA fragment can be determined by running a DNA ladder alongside in the agarose gel
How to separate and differentiate DNA fragments?
Number and position of DNA bands will be determined by the number and size of the DNA fragments
What is the purpose of buffers?
To provide electrical conductivity and maintain pH
Contains EDTA which inhibits the metal dependent nucleases by chelating the divalent cations
What is the function of loading dye?
Tris-HCl - maintains the pH
Glycerol - makes the sample dense so it will sink to the bottom of the well
EDTA - inhibits metal dependent nucleases by chelating the divalent cations
Tracking dye - for visual tracking progression during electrophoresis
Why is tracking dye an important component in the loading dye?
DNA is colourless so some indicators of its movements are required.
DNA bands cannot be observed by naked eyes under normal room light
Why is SYBR safe used instead of ethidium bromide
Ethidium bromide is a carcinogen hence we must be careful when we use it.
SYBR safe is a safer alternative
However, it is more sensitive than SYBR safe dye which results in a more accurate results
How do we know whether individuals are related to one another?
DNA fingerprinting of related individuals will contain many similarly-sized DNA fragments
What does it mean when DNA bands are located at the same location but are of different thicknesses?
DNA fragment is the same
Different thickness due to the different concentration and volume loaded of DNA sample
Why is the same restriction enzyme used?
Ensure that fragments with the same complementary sticky ends are produced so that bonds can form between plasmid and the gene
What if only 1 band is seen in gel electrophoretogram?
Agarose gel concentration is too high, resulting in lesser movement
Restriction enzyme did not recognize any of the restriction site
DNA not cut to generate DNA fragments
- DNA is still in one piece, represented by 1 band
Why does the DNA fragments move to the positive electrode?
DNA is negatively charged hence it gets attracted to positively-charged electrode
Why do smaller DNA fragments move faster than bigger DNA fragments?
Smaller DNA fragments make movement easier through the pores of the agarose molecules.
Larger DNA fragments will get caught among the molecules
