NOTTINGHAM TRENT UNIVERSITY - DNA PROCESSING: STR SEPARATION STUDY NOTES

DNA Processing and STR Separation

Overview of Biological Techniques in Forensic Science

  • Module Code: FORE20007

  • University: Nottingham Trent University

  • Learning Objective: Explain the range of biological techniques available to the forensic scientist.

    • Session Objectives:

    • Utilize knowledge of gel electrophoresis to determine the size of short tandem repeat (STR) fragments.

    • Understand potential errors during electrophoresis and how to rectify them.

Introduction to DNA

  • Key Components of DNA Processing:

    1. DNA Extraction

    2. DNA Quantification

    3. Polymerase Chain Reaction (PCR) & Quantitative PCR (qPCR)

    4. STR Separation (Two Parts)

    • Incorporates three associated labs: online, PCR, and electrophoresis, plus one workshop.

Gel Electrophoresis (GE)

  • Purpose:

    • Separates DNA fragments by size to facilitate DNA profiling.

    • Widely used in biological and forensic laboratories.

Mechanism of Gel Electrophoresis

  • **Process:

    • Post-PCR, DNA fragments are produced and duplicated billions of times.

    • A gel matrix is utilized for separation.

    • DNA is placed in wells of the gel matrix, which is saturated in a buffer solution.

    • An electrical current prompts negatively charged DNA fragments to migrate toward the positive end of the gel.

    • Smaller DNA fragments travel faster through the gel than larger fragments.

    • The bands formed represent groups of identical-sized DNA fragments.

Equipment Setup for Gel Electrophoresis

  • Gel Tank:

    • Houses the gel and buffer.

    • Equipped with a negative cathode (-) and a positive anode (+).

Composition of Gel

  • Materials:

    • Gel made from agarose mixed with buffer (1% - 3%).

    • The concentration of agarose affects separation quality; higher concentrations provide better separation.

    • Nucleic acid stains such as Ethidium Bromide (EtBr, toxic mutagen) and SYBR (safer alternative) can be incorporated to visualize DNA under UV light.

  • Pore Size:

    • Typical pore size of agarose gels: 2000 Å (200 nm) to 100 Å (20 nm).

    • Smaller pores enhance separation abilities.

Buffer Solutions

  • Types of Buffers Used:

    • Tris-acetate-EDTA (TAE)

    • Tris-borate-EDTA (TBE)

  • Functions of Buffer:

    • Maintains experimental pH levels.

    • Provides ions necessary to conduct current across the gel.

Preparation of DNA Samples

  • Loading Dye Addition:

    • DNA samples are combined with a loading dye for visibility and weight.

    • Glycerol serves to provide weight to the DNA sample.

    • Colored dyes assist in tracking samples during gel loading and separation.

Running the Gel

  • Procedure:

    • DNA is loaded into the wells of the gel, then current is applied.

Molecular Dynamics During Electrophoresis

  • DNA Charge Dynamics:

    • The phosphate backbone of DNA contributes H+ ions, imparting a negative charge to the DNA molecule.

    • Consequently, DNA is attracted to the positive charges, moving from cathode (-) to anode (+).

    • The movement facilitated by the ionic current sweeps DNA through the gel.

Result Analysis in Gel Electrophoresis

  • Interpreting Results:

    • Presence of a band indicates the existence of a DNA fragment.

    • Band intensity correlates with DNA concentration:

    • Deeper bands indicate higher concentrations of DNA.

  • DNA Ladder Extra:

    • DNA ladder containing known sized fragments (e.g., 100 bp, 200 bp, and 300 bp) is run alongside samples to provide size comparison.

Questions for Consideration

  • Analyze the results based on visual observations of DNA fragments:

    • Count the number of DNA fragments in sample 2.

    • Determine the heterozygosity or homozygosity of the individual in question.

    • Identify base pairs present in sample 6.

    • Assess size comparison among samples: which has the smallest, largest, and highest concentration DNA fragments?

Broader Applications of Gel Electrophoresis

  • Separation of Other Molecules:

    • Gel electrophoresis can separate various charged molecules including:

    • RNA (different types: tRNA, mRNA)

    • Proteins (enzymes, antibodies)

    • Polypeptides, polysaccharides, and synthetic charged polymers.

SDS-PAGE Technique

  • Description:

    • Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) separates proteins based on mass.

    • Components:

    • SDS: A detergent that denatures proteins and masks the charge of denatured proteins.

    • Polyacrylamide Gel: Allows negatively charged proteins to migrate based on their size.

  • Applications:

    • Protein purification, checking protein purity.

    • Estimating molecular weight to determine protein sizes.

    • Blotting for protein identification.

Errors in Gel Electrophoresis

  • Common Setup Errors:

    • Formation of bubbles.

    • Gel too soft leading to 'warping'.

  • Results Errors:

    • Faint bands, irregular migration patterns, poorly separated bands, and inconclusive results.

Conclusion

  • Gel electrophoresis is a crucial technique employed in forensic science for DNA analysis which requires careful preparation to prevent errors and achieve accurate separation.