Finding Genes and GFP

Finding Genes

• Douglas Prasher aimed to identify a specific gene in jellyfish responsible for green fluorescent protein (GFP) in 1987.
• GFP, a natural protein in jellyfish, converts light energy, causing the jellyfish to glow.
• Prasher hypothesized that GFP could be linked to a protein during its production in a cell, akin to attaching a light bulb.
• To locate the GFP gene, Prasher matched a part of the amino acid sequence of the GFP protein to a genetic code table.
• Base pairing involves Prasher using a complementary base sequence to bind an mRNA matching his prediction.
• A genetic library was screened, which contained thousands of mRNA sequences to discover a perfect match.
• Restriction fragments from the jellyfish genome were separated via gel electrophoresis so that Prasher could find the actual gene that produces GFP.
• Southern blotting is a method named after Edwin Southern, is used determine which fragment contained the actual gene for GFP.

Uses of Restriction Enzymes

• Restriction enzymes allow DNA to be cut from various organisms and people.
• Restriction enzymes enable comparing:
  • Forensics
  • Medical diagnostics
  • Paternity
  • Evolutionary relationships

Comparing Cut DNA

• To compare DNA fragments, they must be separated by size using gel electrophoresis.
• DNA, being negatively charged, moves towards the positive side in an electrical field, and gel electrophoresis is used to separete them.
• Agarose is a gelatin like material DNA is run through

Migration of DNA in Agarose Gel

• Small DNA pieces migrate faster and farther through an agarose gel.
• Large DNA pieces will travel slower and farther behind the smaller ones.

Running a Gel

• DNA is cut with restriction enzymes to create DNA fragments of differing sizes.
• DNA is mixed with a power source in a gel between glass plates
• Longer molecules will appear higher after gel electrophoresis
• Shorter molecules will appear lower ater gel electrophoresis
• Ethidium bromide stains DNA
• DNA fluoresces under UV light when stained with ethidium bromide.

Evolutionary Relationships

• Comparing DNA samples can give clues about evolutionary relationships between different organisms.

Medical Diagnostic

• Comparing normal alleles to disease-causing alleles can help in diagnosing and testing for diseases like Huntington's disease.

Forensics

• Analyzing and comparing DNA samples from crime scenes with those of suspects and victims can help determine perpetrators of crimes.

DNA Fingerprints

• DNA fingerprinting creates unique patterns by comparing DNA banding among individuals
• This method is used to determine the source of blood samples found on a defendant's clothing.

Differences at the DNA Level

• Individual DNA patterns differ due to "junk" DNA sections.
• "Junk" DNA does not code for proteins, but it is made of repeated patterns like CAT and GCC, which vary in the number of repeats.
• The presence of varying numbers of repeats on many sites across the 23 chromosomes contributes to the uniqueness of each person's DNA pattern.

DNA Patterns for DNA Fingerprints

• DNA is cut at specific cut sites, with differing repeats for each person's DNA
• Allele 1 shows the cut sites surrounding repeats of "junk" DNA
• The gel result shows how allele 1 is cut, with the shorter strands travelling further

Differences Between People

• Allele 2 has more repeats in the "junk DNA"
• With different repeats, DNA fingerprint will be different from allele 1

Polymorphisms in Populations

• Differences at the DNA level between people accumulate in "junk" DNA
  • Restriction enzyme in cutting sites shows 2 bands
  • Single base-pair change shows 1 band
  • Sequence duplication shows 2 different bands

Paternity

• Determines who the father is by comparing the DNA

Gel Electrophoresis

• Gel Electrophoresis is a method to separate DNA fragments
• BPB: Bromophenol blue, a tracking dye which is used to track the progress of electrophoresis.
• XC: Xylene Cyanol is another tracking dye

Southern Blot Analysis

• Gel electrophoresis separates DNA fragments produced by restriction enzymes.
• DNA is cut with restriction enzymes.
• Bands on the gel immobilized by blotting onto nitrocellulose paper.
• Radioactive probes bind to fragments with complementary base sequences.
• Probes bind with labeled bands to complete autoradiograph

Changing DNA

• Scientists began wondering if the DNA of a cell could be changed, realizing Frederick Griffith had already accomplished this in bacterial transformation experiments.
• During transformation, a cell uptakes DNA from its surroundings, integrating it into its own genome.
• In Griffith's experiments, live bacteria were transformed by DNA fragments of heat-killed bacteria, changing the live bacteria's characteristics.

Combining DNA Fragments

• Custom-built DNA molecules can be produced in labs and inserted into living cells.
• DNA synthesizers produce short DNA pieces, up to several hundred bases long.
• DNA ligase, an enzyme, is used to join these synthetic sequences to natural ones.
• One organism's gene can be attached to another's DNA.
• Restriction enzymes produce single-stranded overhangs ("sticky ends") in DNA.
• Sticky ends bond with complementary base sequences, thus DNA ligase joins the two fragments.
• Recombinant DNA comprises the resulting molecules.
• Recombinant-DNA technology joins DNA from multiple origins.
• Scientists investigate gene structure and function by manipulating DNA.

Plasmids and Genetic Markers

• Recombinant DNA can use the host cells' DNA replication to copy and replicate at the same time by joining recombinant DNA to a piece of DNA containing a origin replication signal.
• Plasmids are small circular DNA molecules.
• Plasmids replicate newly added DNA with the rest of the cell's genome by joining DNA to a plasmid to transform bacteria.
• Genetic engineering plasmids contain:
  • A replication start signal and origin of replication ("ori")
  • A restriction enzyme cutting site, such as EcoRI
• Yeasts can be transformed with recombinant DNA as well.
• Biologists use artificial chromosomes with centromeres, telomeres, and replication start sites to introduce recombinant DNA into yeast genome.
• When bacteria and target DNA are cut by the same restriction enzymes, with the help of enzymes called restriction enzymes, it can transform the bacteria using recombinant plasmids.
• The new gene combination is returned to a bacterial cell to replicate and produce human growth hormone.
• The recombinant plasmid has a genetic marker (antibiotic resistance gene) that distinguishes plasmid-carrying bacteria from those that don’t.
  • The bacteria culture is treated with an antibiotic after transformation, and only the transformed cells survive due to carrying the resistance gene.