4D: Gel Electrophoresis Flashcards
Gel Electrophoresis
A technique that separates DNA fragments based on their molecular size
- Typically used after a sample of DNA has been cut up using restriction endonucleases or after a PCR - The longer the DNA fragment, the shorter the distance it will travel and vice versa
Standard Ladder
a mixture of DNA fragments of known length that are used to infer the size of fragments in a sample
Kilobase(kb)
a unit of measurement that corresponds to one thousand nucleotides
Process of Gel Electrophoresis
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- DNA samples are placed into the wells using a micropipette alongside a standard ladder
- sits in agarose jelly and immersed in the buffer solution
- An electric current is passed throw the gel using electrodes(negative near wells, positive opposite end)
- as DNA is negatively charged(due to phosphate backbone), it will be attracted to the positive electrode
- means DNA fragments will move from the wells, through the pores of the agarose gel towards the positive electrode
- After a few hours, the current is switched off and the DNA fragments stop moving and settle into bands
- means DNA is now separated by size.
- As DNA is difficult to see with the naked eye, the gel is stained with fluorescent dye(ethidium bromide) allowing the bands to be seen under a UV light.
Base Pair(bp)
a unit of measurement that corresponds to one nucleotide
- Kilobase = one thousand nucleotides
Lane
column of the gel corresponding to each sample of DNA
Band
a line seen in the gel after running gel electrophoresis which corresponds to a collection of DNA fragments of a specific size
- estimate band sizes by comparing to the standadr ladder
- thicker bands means there is more DNA in this band than any others
Electrodes
conductors of electricity which attach to each ends of the agarose gel, allowing an electrical current to pass through
Buffer
an ion-rich solution that carries electrical current through the agarose gel
Variations in distance Travelled in gels is due to
- Voltage: the stronger the electric force, the further DNA will travel
- Gel composition: gels with greater density and agarose concentration increases the difficulty for large fragments to move through
- Buffer concentration: the greater the concentration of ions, the more the electric current is conducted meaning DNA can move further
- Time: the longer the electric current is applied, the further DNA will travel. If too much time passes however DNA may move out of the gel
Genetic Testing
an example of PCR and Gel electrophoresis being applied by screening an individuals DNA for anomalies that may make them susceptible to a particular disease or disorder
- Occurs when an individual possess mutated alleles preventing parts of the body and its cells to function
Example of a genetic disease diagnosed via PCR and Gel Electrophoresis
Cystic fibrosis: a recessive genetic disorder involving the deletion of 3 nucleotides on the CFTR gene
How to test for Cystic Fibrosis
- At birth a routine test called a heel prick is performed in which a blood sample is obtained which then undergoes PCR
- Using specific DNA Primers, which must be complementary to a section of the CFTR gene, join on either side of the mutation so that the sample will only now include the mutated part of the gene
- The DNA sample can then be loaded into the wells of an agarose gel alongside the standard ladder, a sample of a healthy CFTR gene and a sample of a mutated CFTR gene allowing us to compare the results and determine whether the baby either does or does not have Cystic Fibrosis.
- Using specific DNA Primers, which must be complementary to a section of the CFTR gene, join on either side of the mutation so that the sample will only now include the mutated part of the gene
DNA profiling
the process of identification on the basis of an individuals genetic information
- Using DNA profiling we can also determine how related people are as STR’s are found exclusively on autosomal chromosomes meaning the child must inherit half of their parents STR’s
Short Tandem Repeats(STR)
short, repeated sequences of nucleotides found in the non coding regions of nuclear DNA
- 2-5 base pairs long
- Because they are found in non coding regions, they are not affected by natural selection, meaning if two samples of STR in DNA match, we can say with confidence that the samples are from the same person.
