Bio 100 Lab 4 Chapter 4

Introduction to DNA Fingerprinting

Overview of DNA Fingerprinting

DNA fingerprinting is a molecular biology technique used to distinguish between individuals of the same species using samples of their DNA. This method relies on the analysis of specific regions of DNA that vary greatly among individuals, allowing for identification and comparison.

• Purpose: To determine if DNA samples originate from the same or different individuals.

• Applications: Forensics, paternity testing, genetic studies, and biodiversity research.

The Structure of DNA

DNA Molecular Structure

DNA (deoxyribonucleic acid) is a double-helical molecule composed of two strands held together by complementary base pairing. Each strand consists of a backbone made of alternating sugar (deoxyribose) and phosphate groups, with nitrogenous bases attached to the sugars.

• Sugar-Phosphate Backbone: The backbone is identical in all organisms and is formed by covalent bonds connecting deoxyribose sugars to phosphate groups.

• Nitrogenous Bases: Four types: Adenine (A), Thymine (T), Cytosine (C), and Guanine (G).

• Base Pairing: Bases pair specifically via hydrogen bonds: A with T, and C with G.

• Universality: The structure of the sugar-phosphate backbone and base pairing is conserved across plants, animals, and bacteria. Differences between DNA samples arise from the linear sequence of bases, not the chemical structure.

Example: A segment of DNA from a plant, human, and bacterium will have the same backbone structure and base pairing rules, but the order of bases (sequence) will differ.

Restriction Digestion of DNA Samples

Restriction Endonucleases

Restriction endonucleases are enzymes that recognize specific DNA sequences (recognition sites) and cleave the DNA at or near these sites. This process is essential for DNA fingerprinting, as it generates fragments of varying lengths depending on the DNA sequence.

• Discovery: First identified in bacteria by Dr. Werner Arber and Dr. Hamilton Smith in 1968.

• Function: Act as molecular scissors, hydrolyzing the sugar-phosphate backbone at specific sites.

• Types of Cuts: Some enzymes create 'sticky ends' (overhangs), while others produce 'blunt ends' (no overhangs).

Example: EcoRI Restriction Enzyme

• Recognition Sequence: 5'-GAATTC-3'

• Cutting Pattern: EcoRI cuts between G and A, generating sticky ends.

Illustration:

Original DNA:

5'-ATGGAATTCACTAGGCTAC-3' 3'-TACCTTAAGTGATCCGATG-5'

EcoRI cuts at:

5'-G↓AATTC-3' 3'-CTTAA↑G-5'

Resulting fragments:

• Fragment 1: 5'-ATGG | 3'-TACC

• Fragment 2: 5'-AATTCACTAGGCTAC | 3'-GTGATCCGATG

Fragment Size: Expressed in base pairs (bp).

Restriction Enzyme Comparison Table

Additional info: AluI produces blunt ends, which do not have overhanging nucleotides, while EcoRI and MboI produce sticky ends with single-stranded overhangs.

Laboratory Procedure: Restriction Digest

Sample Preparation and Pipetting

Accurate pipetting is essential for preparing DNA samples and enzyme mixes. Adjustable micropipettes are used to measure and transfer small volumes (in microliters, μl).

• Pipette Types: p20 (2-20 μl), p200 (20-200 μl), p1000 (200-1000 μl).

• Proper Technique:

  • Set the correct volume on the dial.

  • Attach a clean tip.

  • Press the plunger to the first stop, immerse in sample, release slowly to draw up liquid.

  • Dispense by pressing to the second stop (blow-out).

  • Change tips between samples to avoid contamination.

Restriction Digest Steps

1. Label tubes for each DNA sample (e.g., Crime Scene [CS], Suspects S1-S4).

2. Add 10 μl of DNA sample to each tube.

3. Add 10 μl of enzyme mix to each tube using a fresh tip for each sample.

4. Mix contents by flicking or tapping, then briefly centrifuge to collect liquid at the bottom.

5. Incubate tubes at 37°C for 45 minutes to allow digestion.

6. Store tubes in the freezer after incubation.

Total Reaction Volume: 20 μl per tube (10 μl DNA + 10 μl enzyme mix).

Analysis of Restriction Digestion

Determining Fragment Number and Size

The number and size of DNA fragments produced depend on the number of recognition sites for the restriction enzyme in the DNA sequence.

• Example (AluI): Recognizes 5'-AGCT-3'. The number of cuts equals the number of recognition sites; the number of resulting fragments is one more than the number of cuts.

• Drawing Fragments: After digestion, the DNA is separated into fragments at each recognition site. The sequence and size of each fragment can be determined by mapping the cut sites.

Comparison of Restriction Enzymes

• EcoRI: Produces sticky ends; only cuts DNA if the recognition sequence is present.

• AluI: Produces blunt ends; cuts at a different recognition sequence.

• MboI: Recognizes 5'-GATC-3'; the number of fragments depends on the number of GATC sites in the DNA.

Restriction Digest Example Table

Additional info: The actual number of cuts and fragments depends on the DNA sequence provided.

Key Definitions and Concepts

• Restriction Endonuclease: An enzyme that cuts DNA at specific recognition sequences.

• Sticky Ends: Single-stranded overhangs produced by staggered cuts in DNA.

• Blunt Ends: Straight cuts with no overhangs.

• Base Pair (bp): A unit of length for DNA fragments, equal to one pair of complementary nucleotides.

• Micropipette: Laboratory instrument used to measure and transfer small volumes of liquid.

Summary

• DNA fingerprinting relies on the unique sequence of bases in DNA and the use of restriction enzymes to generate fragment patterns.

• Restriction enzymes cut DNA at specific sites, producing fragments that can be analyzed to determine genetic similarities or differences.

• Proper laboratory technique, including accurate pipetting and sample handling, is essential for reliable results.