Biotechnology: Detecting Variation Notes

Biotechnology

• Technology based on biology.

• Used for 6000+ years (e.g., making bread and cheese).

• Uses cellular and molecular processes to:

  • Make products (antibiotics, hormones).

  • Modify genes, treat genetic diseases (DNA manipulation).

  • Create alternative fuel sources (fermentation, enzymes).

  • Improve agriculture (enhance nutrient content, reduce pesticide use).

DNA Extraction

• Needed to sequence DNA.

• Lysis buffer:

  • Contains detergents that rupture cell and nuclear membranes, releasing DNA.

• Enzymes:

  • Cut apart histones (proteins) to free DNA.

• Salt solution:

  • Clumps cellular debris from DNA.

• Isopropyl alcohol:

  • Brings DNA out of solution, making it visible.

Restriction Enzymes

• Found in bacteria.

• Part of their immune system, defending against viruses and plasmids.

• Considered static or fixed immunity.

• Cut DNA at specific locations (molecular scissors).

• Recognize and cut palindromic sequences.

  • Palindrome examples: LEVEL, MADAM.

  • DNA palindrome are specific base sequences

• Over 600 restriction enzymes identified; each cuts DNA at specific base sequences.

• Exposed nitrogenous bases that overhang are called ‘sticky ends’.

Gel Electrophoresis

• Used to separate DNA fragments or other molecules.

• Separates molecules based on size; longer molecules move slower, shorter molecules move faster.

Restriction Fragment Length Polymorphisms (RFLPs)

• DNA is cut into fragments using restriction enzymes.

• RFLPs are fragments of varying lengths and is a type of genetic fingerprinting.

• RFLPs account for the differences in DNA fragments seen with gel electrophoresis.

• Analysis of RFLP variation was vital in genome mapping and genetic disease analysis.

  • To determine the chromosomal location of a disease gene, analyze DNA from afflicted family members and look for RFLP alleles with inheritance patterns similar to the disease.

  • RFLP analysis could reveal who is at risk or likely to be a carrier of mutant genes.

DNA Replication

• Copying DNA.

• Key components: Helicase, RNA Polymerase, DNA Polymerase, Ligase.

Polymerase Chain Reaction (PCR)

• Rapidly copies a specific DNA sequence.

• Cycles:

  • Heat to denature DNA.

  • Cool to allow primers to bind.

  • Warm to copy sequence using DNA polymerase.

Sanger Method - Dideoxy Chain Termination

• Uses a similar mechanism to DNA Replication but using dideoxynucleotides (ddNTPs).

• ddNTPs lack a 3’ oxygen group, stopping replication, and are fluorescently labeled.

• Creates multiple length fragments that are run through gel electrophoresis.

• A sensor picks up each fragment's last nucleotide.

• Process:
  $\text{DNA (template strand) } 5' \rightarrow 3'$
  $\text{Primer } 3' \rightarrow 5'$
  $\text{DNA polymerase}$
  $\text{Deoxyribonucleotides}$
  $\text{Dideoxyribonucleotides (fluorescently tagged)}$

DNA Polymorphisms

• The average two people share about 99.9% of their DNA.

• Variations in DNA sequence between individuals are termed "polymorphisms".

• Sequences with the highest degree of polymorphism are very useful for DNA analysis.

Short Tandem Repeats (STRs)

• DNA polymorphisms where different numbers of copies of a repeat element occur.

• Short sequences (2-5 base pairs) repeated numerous times in a head-tail manner (e.g., "gatagatagatagata" is 4 copies of "gata").

• DNA STRs are like beads on a string, with the number of beads differing for each person.

• Diploid organisms inherit 23 chromosomes from each parent, including their STRs.

• Scientists examine many different STR locations to differentiate individuals.

• The FBI and the police use thirteen different STRs in a standard DNA test, known as Combined DNA Index System (CODIS).

• Everyone has two of each of these STRs, so that makes 26 different copy numbers that make up a person’s CODIS signature.

  • The chance that you and an unrelated person have the exact same CODIS signature is less than one in a .

RFLPs vs STR

• Both have been useful for identification of samples retrieved from crime scenes, in the determination of paternity, and in the characterization of genetic diversity or breeding patterns in animal populations; however, STRs are usually the preferred method nowadays.

• Gel electrophoresis is used in both cases, and probes highlight the varying fragments.

• There is usually less variation among individuals RFLPs than STRs.

• RFLPs usually require a large amount of DNA to start; whereas STRs use PCR to amplify small samples of DNA.

• STR analysis does not cut the DNA with restriction enzymes.