SAC 1? - gel electrophoresis Flashcards
(12 cards)
After restriction enzymes have cut DNA into different fragment sizes, they need to be sorted and viewed. How does gel electrophoresis do this?
Gel electrophoresis sorts DNA fragments by size.
An electric current pulls the negatively charged DNA through a gel - smaller fragments move faster and farther, so they separate by length and can be viewed as bands.
What is the role of the agarose gel?
- submerged in a buffer solution (an ion-filled solution that carries the electrical current through).
- the agarose molecules re held by hydrogen bonds, forming tiny pores
- DNA in the wells migrates through the pores to the positive electrode
What is the role of the electrical current?
- DNA in the wells migrates its way through the pores to the positive electrode as it is negatively charged
- then the gel van be examined to identify number and size of DNA fragments in each sample
Is DNA charged? If so, how does this affect its movement through the electrical field?
Yes, negatively charged.
This means it is attracted to positive charges, moving down towards the end
Which fragment sizes will travel the furthest through the gel?
Smaller fragments move faster and therefore further through the gel as they can fit through the pores more than longer fragments
Other than fragment size, what factors can affect how far DNA fragments travel during gel electrophoresis?
- Voltage (stronger force/higher voltage = faster and further DNA travels)
- Gel composition (greater density/concentration = more difficult for larger fragments to move thru)
- Buffer concentration (greater concentration of ions = more electric current conducted = DNA moves further)
- Time in gel (longer electric current applied = further DNA travels)
What is the role of the buffer?
- agarose gel is submered in buffer solution
- carries the electrical current through the agarose gel
- maintains temperature so gel does not melt with current
What is the role of the known markers or DNA ladder?
- set of DNA fragments with known lengths that will be used to compare the unknown DNA fragments to
- loaded into one well
- vital as DNA fragments of the same size don’t always travel the same distance on all gels
applications of PCR and gel electrophoresis
- diagnosis of genetic disorders (genetic testing)
- identification of a suspect in a crime (forensic testing)
- to determine paternity (and maternity)
- DNA profiling to distinguish different species
genetic/DNA testing
- used to identify changes in DNA sequences or chromosome structure
- results of a genetic test can confirm a suspected genetic condition, or determine a person’s chance of developing or passing on a genetic disorder eg. cystic fybrosis, huntington’s disease
- genetic disorders result from someone having mutated alleles -> can be as small as a single base change
cystic fybrosis
- RECESSIVE genetic disorder
- deletion of 3 nucleotides on the CFTR gene
- both parents can be carriers (homozygous recessive)
- child needs 2 copies of mutated CFTR gene
- results in mucus in organs like lungs, difficulty breathing, shorter lifespan
- blood sample extracted using heel prick
DNA profiling
- a technique by which individuals can be identified and compared via their respective DNA profiles.
-within introns (non-coding) exists SHORT TANDEM REPEATS (STRs) - stretches of DNA made up of repeating sequences - each individual has 2 alleles for each STR, but different numbers of repeats at a DNA locus
- not affected by natural selection (not used to code for proteins)
-> have higher mutation rate and show GREATER VARIATION between individuals without affecting functioning
->STRs in 2 samples match = belong to same person
