Lab Notes

Fall Biology

Table of contents

Introduction to Biotechnology Lab

DNA gel electrophoresis was developed in the 1970s and hsa been important in bringing advances in biotechnology.
In activity 1:
- Explore restrictive enzyme digestion, polymerase chain reaction (PCR), and gel electrophoresis in a simualtion investigating sickle cell mutations.
In activity 2:
- Use a mini gel setup to electrophorese dyes across an agarose medium.

Scientists can analyze genetic variations to predict relationships between groups or individual.
- Sometimes referred to as ‘DNA profiling’ or ‘DNA fingerprinting’.
Is used to match an individual to a DNA sample.

Sickle cell anemia is a genetic disease caused by a point mutation in the beta-globin gene.
- Switches one adenine nucleotide to thymine.
- Changes glutamic acid to valine. Alters shape of hemoglobin.
Cariers have one single sickle cell allelle and one wild-type allele.
- Health risk for people with one mutated allele is minor.

DNA typing is useful for diagnosing individuals.
- Location and sequence of the sickle cell point mutation is known.
DNA typing can determine if an individual is homozygous wild-type, heterozygous, or homozygous for the sickle cell mutation.

To perform DNA typing, a sample is collected from the individual.
DNA is found in the nucleus of every human cell, from skin, hair, saliva, blood,e tc.
Each sample contains the entire human genome.
- Only a small region of the beta-globin gene is of interest.
It is not possible to detect the mutation from a single copy of DNA.
- The desired region must be copied over and over in a process called amplification to determine if the mutation is present.
PCR amplifies the region of interest (the region known to contain the mutation).
- After PCR is completed, there will be millions of copies of that region of DNA.
Leads to exponential amplification of the DNA region of interest.
- Doubles the number of copies of the region during every cycle.
Initial sample containing two copies of DNA sequence amplified through 35 cycles of PCR yields 2 to the 35 copies of the sequence.

PCR reaction requires template DNA, free nucleotides (dNTPs), sequence-specific DNA primers, DNA polymerase, water, and salts.
Cycle:
1. Denaturation. DNA is heated to near boiling so the two strands separate.
2. Annealing. DNA primers (usually 18-22 bases long) are designed and synthesized in a laboratory. Primers bind to their complementary sequences as their temperature is lowered.
  - One primer binds to each DNA strand, and each primer is the reverse complement of the DNA strand to which it binds. Region between the two primers is the region being amplified.
3. Extension. The temperature is raised again; DNA synthesis occurs as the DNA polymerase adds nucleotides to the 3’ end of each primer. DNA polymerase is an enzyme that catalyzes the DNA synthesis reaction by adding free nucleotides to the DNA strand. Primers are extended to become the complement strands.
4. Amplification.Once extension is completed, the process of denaturing, annealing, and extension is repeated several times.

Next step in DNA profiling is to visualize the PCR products (amplified DNA sequence) with electrophoresis).
Sequence of the region of interest for beta globin is known.
However, a different region may have been amplified.
Running PCR products on a gel verifies that the region was properly amplified.

Each DNA sample is pipeted into a small well.
DNA has a negative electrical charge.
- When an electric current is passed through the gel, DNA migrates towards the anode (positive pole).
DNA fragments travel through gels at speeds inversely related to their size.
- When current stopes, fragments of DNA remain at different places inside the gel.
- DNA fragments of the same size appear as bands in the rectangular shape of the well.
DNA fragments separated by size on the gel can then be visualized by staining.
- DNA can be stained with a fluorescent dye that requires UV light for visualization.
Appoximate size of the DNA fragments in each band can be determined by comparing the distance travelled b the band to a DNA ladder composed of DNA fragments of known sizes running in the same gel.
- band with DNA fragments should match the sizee of the region of interest can be extracted for testing.

In the simulation, amplified fragments of DNA contain a region known to have the sickle cell mutation.
Unknown if the mutation is present or absent.
Nucleotides are too small ti visualize.
Restriction endonucleases (restriction enzymes) are enzymes that act like tiny scissors to cut DNA at specific nucleotide sequences.
Scientists collect enzymes from bacteria and use them for DNA profiling because the enzymes digest animal DNA.
Some restriction enzymes cut specific DNA sequences so that all the nucleotides are still bound to complementary bases.
- Ends of DNA strands are called ‘blunt ends’.
- Uneven strands of DNA where not all nucleotides are bound to DNA strands are ‘sticky ends’.

The next step in DNA typing is visualizing results of the nucleotide restriction of the PCR-amplified DNA sequence by gel electrophoresis.
Number bands and sizes indicate presence or absence of restriction sites in amplified DNA.
If there are two distinct bands, then we can assume there was one restriction site and the estriction enzyme used made one cut.