DNA Processing and STR Separation via Gel Electrophoresis

The study of Short Tandem Repeat (STR) separation is a critical component of the module FORE20007: Biological Techniques in Forensic Science.
Session objectives include:
- Utilizing an understanding of gel electrophoresis to determine the size of STR fragments.
- Examining how errors occur during the process of electrophoresis.
- Identifying methods to resolve common electrophoresis errors.
The broader DNA processing pipeline involves the following steps:
1. DNA Extraction
2. DNA Quantification
3. PCR & qPCR (Polymerase Chain Reaction and quantitative PCR)
4. STR Separation I
5. STR Separation II
The curriculum includes three associated laboratories: Online, PCR, and Electrophoresis, along with one dedicated workshop for STR Separation I and II.

Post-PCR Status:
- After the PCR process, specific fragments of DNA are obtained.
- Each target fragment has been copied over a billion times.
Mechanism of Action:
- Gel electrophoresis is the technique used to separate these DNA fragments based specifically on their size.
- It is a vital tool for separating STRs for the purpose of DNA profiling.
- Standard gel electrophoresis is used in general biological laboratories, while amended versions of the technique are utilized in both biological and specialized forensic labs.
General Procedure:
- DNA is loaded into "wells" (holes) within a gel matrix that is submerged in a buffer solution.
- An electrical current is applied to the system.
- Because DNA carries a negative charge ( $-$ ), the fragments are forced to move toward the positive end ( $+$ ).
- The gel matrix acts as a sieve; small fragments move through the matrix faster than large ones.
- Bands: Each visible band in the gel represents a group of DNA fragments that are the same size.
- Standards: DNA fragments of known sizes (a ladder) are run alongside the samples to provide a reference for size comparison.

Gel Tank:
- This is the environment where the electrophoresis performance occurs.
- It houses both the gel and the buffer solution.
- It features two electrodes: a negative cathode ( $-$ ) and a positive anode ( $+$ ).
The Gel Matrix:
- DNA fragments migrate through this matrix.
- Construction: It is typically a combination of agarose and buffer, melted and poured to set.
- Concentration: Agarose concentration likely ranges between $1\%$ and $3\%$ .
- Effect of Concentration: The specific percentage of agarose depends on the required level of separation. A higher percentage ( $\%$ ) results in better separation of fragments.
- Nucleic Acid Stains: These are often incorporated into the gel. They bind to DNA and make it visible under alternative light sources, such as Ultraviolet (UV) light.
- Stain Options:
  - Ethidium Bromide (EtBr): A toxic mutagen.
  - SYBR: A safer alternative for DNA staining.
Comparison of Gel Types and Pore Sizes:
- Agarose Gels: Feature larger pores of approximately $2000\,\text{\AA}$ ( $200\,nm$ ).
- Polyacrylamide Gels: Feature smaller pores of approximately $100 - 200\,\text{\AA}$ ( $20\,nm$ ).
- Numerical Note: An Angstrom ( $\text{\AA}$ ) is a unit of length equal to one hundred-millionth of a centimeter, or $10^{-10}\,m$ .
- Principle: The smaller the pores in the gel, the better the ability to separate fragments precisely.

Buffer Solutions:
- Once the gel solidifies, the buffer is poured over it.
- Common types include Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
- Purpose: Maintains the pH of the experiment and provides the ions necessary to carry the electrical current through the gel.
- Ion Flow: Ions move through the gel from the negative cathode to the positive anode, and this current "sweeps" or pushes the DNA fragments along.
DNA Samples and Loading Dye:
- Pipetting a liquid into a buffer-filled well is difficult because the sample may float away.
- Loading dye is added to DNA samples to address this.
- Glycerol: Added to provide weight, ensuring the DNA sinks into the well.
- Coloured Dyes: Added for visibility, allowing the scientist to see the sample in the well and track its progress during separation with the naked eye.

Charge Status: The phosphate groups in DNA release $H^+$ ions in buffer solutions, rendering the DNA molecule negatively charged.
Movement Dynamics:
- As a negatively charged molecule, DNA is attracted to positive charges.
- It moves away from the negative cathode ( $-$ ) and toward the positive anode ( $+$ ).
- Smaller fragments move through the pores of the matrix more quickly than larger ones.
Visualization Systems:
- The loading dye allows for tracking migration with the naked eye during the run.
- The nucleic acid stain (e.g., SYBR) allows the actual DNA bands to be resolved under UV light after the run.

Interpreting Bands:
- The presence of a band indicates a DNA fragment.
- The intensity and depth of the band correspond to the concentration of the DNA fragment.
DNA Ladders:
- A DNA ladder is run simultaneously with samples.
- It consists of DNA fragments of known sizes (for example: $100\,bp$ , $200\,bp$ , $300\,bp$ , etc.).
- By comparing sample bands to the ladder, the general size of the fragments can be determined.

Question: Can gel electrophoresis be used for other molecules? Yes. It can separate RNA (all species like tRNA, mRNA), proteins, enzymes, antibodies, charged molecules, peptides, polysaccharides, and synthetic charged polymers.
SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis):
- Purpose: Separates proteins by mass.
- Sodium Dodecyl Sulphate (SDS): A detergent that denatures proteins and acts as a surfactant, masking the inherent charge of the denatured protein with a uniform negative charge.
- Polyacrylamide Gel: In this matrix, negatively charged proteins move faster if they are smaller.
- Applications:
  - Protein purification: To check the purity of protein samples.
  - Molecular weight estimation: To determine the size of proteins.
  - Protein identification: Used in techniques like blotting.

Setting Up Errors:
- Bubbles: Can disrupt the uniform flow of current and distort bands.
- Gel too soft: Leads to difficulty in handling and poor well integrity.
- Warping: Physical distortion of the gel matrix.
Results Errors:
- Faint bands: Caused by low DNA concentration or poor staining.
- Poorly separated bands: Often due to incorrect gel concentration (agarose $\%$ ) or running the voltage too high/too long.
- Irregular migration patterns: Can be caused by buffer exhaustion, incorrect pH, or contaminants in the sample.