DNA profiling, PCR and electrophoresis

Introduction to DNA Profiling

Imagine you are a forensic scientist at a crime scene. You find a single hair or a tiny drop of blood. How do you turn that microscopic bit of evidence into proof that identifies a suspect? The answer lies in DNA profiling. In this chapter, we will explore how scientists use two incredible technologies—PCR and electrophoresis—to create a "genetic fingerprint" that is unique to every individual (except identical twins!).

This topic is part of your study on Topic 6: Immunity, Infection and Forensics. While other chapters look at how a body decomposes, this chapter focuses on the high-tech laboratory side of solving mysteries and proving genetic relationships.

What is DNA Profiling?

While \(99.9\%\) of human DNA is exactly the same in everyone, there are sections of "non-coding" DNA (DNA that doesn't code for proteins) that vary hugely between people. These sections contain Short Tandem Repeats (STRs).

STRs are short sequences of bases (e.g., GATA) that repeat over and over again. The number of times these sequences repeat at a specific locus (position on a chromosome) varies from person to person. Because we inherit one chromosome from each parent, we have two sets of these repeats for every locus. DNA profiling works by looking at these specific areas to see how many repeats a person has.

Key Takeaway:

DNA profiling doesn't look at your whole genome; it looks at specific STRs in the non-coding regions of your DNA to create a unique pattern of fragments.

Step 1: The Polymerase Chain Reaction (PCR)

In forensics, you often start with a very small sample of DNA. To analyze it, you need millions of copies. PCR is essentially a "molecular photocopier" that amplifies a specific segment of DNA.

What you need for PCR:

1. The DNA Sample: The "template" you want to copy.
2. DNA Polymerase: Specifically Taq polymerase, which is an enzyme that can withstand high heat.
3. Primers: Short pieces of DNA that tell the polymerase where to start copying.
4. Free Nucleotides: The raw building blocks (\(A, T, C, G\)) to build the new DNA strands.
5. Buffer: To keep the \(pH\) stable for the enzymes.

The Three Stages of PCR:

PCR happens in a machine called a thermal cycler, which changes temperature in a cycle:

1. Denaturation (approx. \(95^\circ C\)): The DNA is heated to break the hydrogen bonds between the two strands, separating them into single strands.
2. Annealing (approx. \(55^\circ C\)): The mixture is cooled so that the primers can bind (anneal) to the beginning of the STR sequences on the single-stranded DNA.
3. Extension (approx. \(72^\circ C\)): The mixture is heated again. The Taq polymerase lines up free nucleotides alongside the template strand, starting from the primers, to create a double-stranded DNA molecule.

Don't worry if this seems tricky at first! Just remember: Heat to separate, Cool to bind, Warm to build. This cycle is repeated about \(30\) times. Because the amount of DNA doubles every time, the growth is exponential: \(2^n\), where \(n\) is the number of cycles.

Step 2: Gel Electrophoresis

Once you have amplified the DNA using PCR, you have a mixture of DNA fragments of different lengths. You need to separate them to see the profile. This is done using Gel Electrophoresis (Core Practical 14).

How it Works:

1. DNA fragments are placed into wells in a slab of agarose gel.
2. The gel is covered in a buffer solution, and an electric current is passed through it.
3. DNA is negatively charged (due to the phosphate groups). This means it will move towards the positive electrode (the anode).
4. The gel acts like a sieve. Smaller DNA fragments can move through the holes in the gel more easily and quickly than larger fragments.

The Result:

Over time, the fragments separate based on their size. The smallest fragments will be furthest from the wells, and the largest fragments will be closest to the wells. Scientists then use a fluorescent dye or radioactive markers to make the DNA "bands" visible.

Applications of DNA Profiling

Once the electrophoresis is complete, you get a pattern of bands. This can be used for:

1. Forensic Identification

To identify a suspect, their DNA profile must be an identical match to the DNA profile found at the crime scene. Every single band must align perfectly.

2. Genetic Relationships (Paternity and Phylogeny)

We inherit half our DNA from our mother and half from our father. Therefore, in a DNA profile:
- Every band in a child's profile must be present in either the mother's or the father's profile.
- This can also be used in animals and plants to study genetic relationships and how closely related different species are (this relates to genomics and proteomics mentioned in section 5.18).

Common Mistakes to Avoid

- Mixing up the electrodes: Remember, DNA is Negative, so it moves toward the Positive electrode (the Anode). Mnemonic: DNA is "Negative Nancy" walking toward "Positive Paul."
- Confusing PCR and Electrophoresis: PCR makes the DNA (amplification); Electrophoresis sorts the DNA (separation).
- Reading the Gel wrong: Remember that the smallest fragments travel the furthest. If a band is right at the bottom of the gel, it represents a very short sequence of DNA.

Quick Review Box

STRs: Unique repeating sequences in non-coding DNA.
PCR: Amplifies DNA using cycles of heating (\(95^\circ C\)), cooling (\(55^\circ C\)), and warming (\(72^\circ C\)).
Taq Polymerase: Heat-stable enzyme used in PCR.
Electrophoresis: Separates DNA by size using an electric current.
Anode: The positive electrode that DNA moves toward.

Did you know? Taq polymerase was originally discovered in a bacteria called Thermus aquaticus, which lives in hot springs and hydrothermal vents! This is why it doesn't denature at the high temperatures used in PCR.