SAC 1? - gel electrophoresis

Question 1

After restriction enzymes have cut DNA into different fragment sizes, they need to be sorted and viewed. How does gel electrophoresis do this?

Answer 1

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.

Question 2

What is the role of the agarose gel?

Answer 2

• 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

Question 3

What is the role of the electrical current?

Answer 3

• 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

Question 4

Is DNA charged? If so, how does this affect its movement through the electrical field?

Answer 4

Yes, negatively charged.
This means it is attracted to positive charges, moving down towards the end

Question 5

Which fragment sizes will travel the furthest through the gel?

Answer 5

Smaller fragments move faster and therefore further through the gel as they can fit through the pores more than longer fragments

Question 6

Other than fragment size, what factors can affect how far DNA fragments travel during gel electrophoresis?

Answer 6

• 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)

Question 7

What is the role of the buffer?

Answer 7

• agarose gel is submered in buffer solution
• carries the electrical current through the agarose gel
• maintains temperature so gel does not melt with current

Question 8

What is the role of the known markers or DNA ladder?

Answer 8

• 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

Question 9

applications of PCR and gel electrophoresis

Answer 9

• 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

Question 10

genetic/DNA testing

Answer 10

• 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

Question 11

cystic fybrosis

Answer 11

• 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

Question 12

DNA profiling

Answer 12

• 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