The use of labelled DNA probes that can be used to locate specific genes by complementary base pairing (DNA hybridisation). The use of these techniques in medical diagnosis. The principles of DNA sequencing and the development of high-throughput sequencing. Genetic fingerprinting and its use in determining genetic relationships and the genetic variability within a population, based on variable number tandem repeats (VNTRs), separated by size using gel electrophoresis.

What this dot point is asking

AQA wants you to explain how labelled DNA probes locate specific sequences by complementary base pairing (hybridisation), how this is used in medical diagnosis, the principle of DNA sequencing and high-throughput sequencing, and how genetic fingerprinting uses VNTRs separated by gel electrophoresis to compare individuals.

Labelled DNA probes and hybridisation

To find a specific gene or allele:

1. Make a probe whose base sequence is complementary to the target sequence, and attach a label (radioactive, so it can be detected on film, or fluorescent, so it glows under UV light).
2. Make the sample DNA single-stranded (by heating) and mix it with the probe.
3. Where the target sequence is present, the probe hybridises to it by complementary base pairing.
4. Detect the label. A signal means the target sequence is present; no signal means it is absent.

Use in medical diagnosis

DNA probes allow doctors to test for disease-causing alleles before symptoms appear:

• Identifying inherited conditions such as those caused by specific alleles (a probe complementary to the faulty allele binds only if the person carries it).
• Genetic screening and counselling, so couples can know the chance of passing on a disorder.
• Personalised medicine. Probes (and sequencing) can reveal which alleles a patient has, so the most effective drug and dose can be chosen, and drugs likely to cause harmful side effects can be avoided.
• Probes for many alleles can be fixed to a DNA microarray, testing for hundreds of alleles at once.

Principles of DNA sequencing

DNA sequencing determines the exact order of bases in a length of DNA. The classic (chain-termination) method works by:

• Making many copies of the DNA, each terminated at a different point by a modified, labelled nucleotide.
• Separating the fragments by size (electrophoresis), so the labelled end of each tells you which base is at that position.
• Reading the sequence off in order.

High-throughput (next-generation) sequencing sequences millions of fragments at once, in parallel and automatically. This has made sequencing far faster and cheaper: a whole human genome that once cost billions of dollars and took years can now be sequenced quickly for a few hundred dollars. This supports genome projects, comparison of species and personalised medicine.

Genetic fingerprinting

The non-coding DNA between genes contains variable number tandem repeats (VNTRs): short base sequences repeated many times. The number of repeats at each VNTR locus varies greatly between individuals, so the lengths of these regions are almost unique to each person (except identical twins).

How a genetic fingerprint is made:

1. Extract DNA from a sample (blood, saliva, hair root) and amplify it if necessary using PCR.
2. Cut the DNA with restriction endonucleases at sites around the VNTRs, producing fragments whose length depends on the number of repeats.
3. Separate by size using gel electrophoresis: DNA is placed in wells in a gel and a voltage is applied; DNA is negatively charged so it moves toward the positive electrode, and smaller fragments move further. (The fragments are made single-stranded and transferred to a membrane.)
4. Visualise the fragments using labelled probes, giving a pattern of bands like a barcode.

Uses of genetic fingerprinting

• Determining genetic relationships. Every band in a child's fingerprint must come from one parent. This is used in paternity and maternity testing and to establish family relationships.
• Forensic science. A suspect's fingerprint is compared with DNA found at a crime scene; matching banding patterns indicate the DNA could be from the same person.
• Measuring genetic variability within a population. Comparing fingerprints across many individuals shows how much variation exists, which is useful in conservation and in studying populations.

Common mistakes

Try this

Q1. Explain why a DNA probe must be single-stranded to detect a specific allele. [2 marks]

• Cue. Only single-stranded DNA can base-pair (hybridise) with the complementary single-stranded target; if the probe were double-stranded, its bases would already be paired and could not bind the target.

Q2. Explain why VNTRs are more useful than genes for identifying individuals. [2 marks]

• Cue. The number of repeats at VNTR loci varies greatly between individuals, giving almost unique fragment lengths; genes are far more similar between people, so they would not distinguish individuals.

Q3. Describe how gel electrophoresis separates DNA fragments. [3 marks]

• Cue. DNA fragments are placed in wells in a gel; a voltage is applied; DNA is negatively charged so it moves toward the positive electrode; smaller fragments move faster and further, separating the fragments by size.