Training Brochure

1

Sample Collection & Preservation

Understanding Biological Samples

1. Blood, saliva (buccal swabs), semen, hair, bone, and tissue samples.
2. Sample handling techniques to avoid contamination.

Collection Methods

1. Post-Mortem sample collection and storage.
2. Sample collection from living subjects.

Preservation & Storage

1. Use of sterile collection kits.
2. Temperature-controlled storage methods.
3. Sample labeling and documentation for traceability.

Training Focus: Hands-on practice with collection kits, labeling, and documentation.

2

DNA Extraction

Extraction Methods

1. Organic (Phenol-Chloroform).
2. Automatic DNA Extraction.
3. Silica column-based extraction.
4. Magnetic bead-based purification.

Steps of DNA Extraction

1. Lysis: Breaking open cells to release DNA.
2. Protein Removal: Eliminating proteins using organic solvents or protease treatment.
3. Purification: Using alcohol precipitation or column-based methods.
4. Storage: Storing extracted DNA at -20°C to -80°C.

Training Focus: Hands-on extraction of DNA from biological samples.

3

DNA Quantification

Purpose of Quantification

1. Assessing DNA quality and quantity before PCR.
2. Ensuring optimal DNA input for amplification.

Techniques Used

1. Spectrophotometry (NanoDrop): Measures DNA concentration based on absorbance at 260/280 nm.
2. Agarose Gel Electrophoresis: Uses EtBr to quantify DNA.
3. Quantitative PCR (qPCR): Determines human-specific DNA quantity for STR profiling.

Training Focus: Practical experience using NanoDrop, Gel Electrophoresis, and qPCR for DNA quantification.

4

Polymerase Chain Reaction (PCR) Amplification

Principles of PCR

1. Denaturation, Annealing, and Extension.
2. Multiplex PCR for amplifying multiple STR loci.

STR Kits Used

1. 27 STR Marker Kit.

PCR Setup

1. DNA template, primers, buffer, dNTPs, polymerase.
2. Thermal cycling conditions for STR amplification.
3. PCR contamination control.

Training Focus: PCR setup, reagent preparation, and thermal cycling.

5

Capillary Electrophoresis (CE) & STR Analysis

Introduction to Capillary Electrophoresis

1. Separation of DNA fragments based on size.
2. Use of fluorescent dyes for STR detection.

Equipment Used

1. ABI 3500/3500xL Genetic Analyzer.
2. Sample preparation and loading.

Data Analysis

1. Interpretation of electropherograms.
2. Identifying peaks, stutter, artifacts, and allelic dropout.

Training Focus: Operating CE instruments and analyzing STR profiles using GeneMapper software.

6

STR Data Interpretation & Quality Control

Understanding Electropherograms

1. Peak height ratios, allelic ladders, and stutter analysis.
2. Interpretation of homozygous vs. heterozygous peaks.

Ensuring Data Accuracy

1. Internal positive and negative controls.
2. Addressing contamination and drop-in/drop-out artifacts.

DNA Database

1. UMID-AIIMS DNA Database.

Training Focus: Comparative analysis of known and unknown STR profiles.