Recombinant DNA Technology (AQA A-Level Biology): Revision Notes
Genetic Fingerprinting
Genetic fingerprinting is a powerful diagnostic technique widely used across forensic science, medical diagnosis, and plant and animal breeding. The method works because every individual's DNA is unique (except for identical twins), creating a distinctive pattern that can be used for identification and comparison.
The uniqueness of DNA makes genetic fingerprinting one of the most reliable identification methods available to science, with applications extending far beyond criminal investigations into medical diagnosis and conservation efforts.
The scientific foundation
Variable number tandem repeats (VNTRs)
The technique exploits a key feature of eukaryotic genomes: most DNA does not code for proteins. Approximately 95% of human DNA is non-coding, including sequences called Variable Number Tandem Repeats (VNTRs).
VNTRs are repetitive DNA sequences where the number and length of repeats differs between individuals. Even closely related people show variation in their VNTR patterns, though the similarity increases with genetic relatedness. The probability of two unrelated individuals sharing identical VNTR sequences is extremely low, making these sequences ideal for creating unique genetic profiles.
Understanding VNTRs
Think of VNTRs as genetic "stutters" - short sequences that repeat over and over, but the number of repetitions varies between people. It's like having different people say "ha-ha-ha" but one person says it 5 times, another 12 times, and another 8 times. This variation creates the unique patterns used in genetic fingerprinting.
Gel electrophoresis technique
Gel electrophoresis separates DNA fragments according to size using an electric current. DNA fragments are loaded onto an agar gel and voltage is applied across the gel. The gel's resistance means larger fragments move more slowly, while smaller fragments travel further during the same time period.
For genetic fingerprinting, DNA fragments must be cut into manageable sizes using restriction endonucleases. These enzymes can only sequence DNA fragments up to approximately 500 bases long, so larger genes and whole genomes require fragmentation before analysis.
When fragments are labelled with radioactive probes, their final positions can be determined by placing X-ray film over the gel. The radioactivity exposes the film, showing exactly where each fragment is located.
Critical Concept: Fragment Size and Movement
Remember that in gel electrophoresis, there's an inverse relationship between fragment size and distance travelled. Smaller fragments can move more easily through the gel matrix, while larger fragments encounter more resistance. This principle is fundamental to understanding genetic fingerprint patterns.
The five-stage process
The Five Essential Stages
Genetic fingerprinting follows a precise five-stage process that must be completed in order. Each stage builds on the previous one, and the quality of the final result depends on careful execution of every step.
1. Extraction
Even tiny biological samples (blood drops, hair roots) contain sufficient DNA for fingerprinting. The first step involves extracting and purifying DNA from the sample. Since DNA quantities are often small, the polymerase chain reaction can amplify the available material.
2. Digestion
The extracted DNA is cut into smaller fragments using restriction endonucleases. These enzymes are selected for their ability to cut near, but not within, the target VNTR sequences.
3. Separation
DNA fragments are separated by size using gel electrophoresis. After separation, the gel is immersed in alkali to separate double-stranded DNA into single strands, preparing them for the next stage.
4. Hybridisation
Radioactive DNA probes (or fluorescent alternatives) are added to bind specifically with VNTR sequences. These probes have complementary base sequences to the VNTRs and attach under controlled conditions of temperature and pH. Different probes target different VNTR sequences.
5. Development
The gel is covered with a nylon membrane, then X-ray film is placed over it. Radiation from the radioactive probes exposes the film, creating a pattern of dark bands. Each band represents the position of a DNA fragment containing VNTR sequences. This banding pattern is unique to each individual (except identical twins).
Worked Example: Memory Aid for the Five Stages
Use the mnemonic: "Every DNA Sample Has Distinctive patterns"
- Extraction - Get the DNA out of the sample
- Digestion - Cut DNA into fragments with restriction enzymes
- Separation - Sort fragments by size using gel electrophoresis
- Hybridisation - Attach radioactive probes to VNTR sequences
- Development - Create the final banding pattern on X-ray film
Interpreting results
Results are analysed by comparing banding patterns between samples. Automated scanning machines measure fragment lengths and calculate the probability of matches. The closer the match between two patterns, the higher the likelihood that the DNA samples come from the same person.
Key points for interpretation:
- Smaller DNA fragments travel further in gel electrophoresis
- VNTR inheritance follows normal genetic patterns but doesn't affect phenotype
- Pattern comparison requires statistical analysis to determine match probability
Statistical Analysis in Genetic Fingerprinting
Genetic fingerprinting doesn't provide absolute proof of identity - it provides statistical probability. The results are expressed as odds, such as "the probability that this DNA came from someone other than the suspect is 1 in 10 million." Understanding this statistical nature is crucial for proper interpretation.
Applications
Genetic relationships and paternity
Genetic fingerprinting can establish family relationships since individuals inherit half their genetic material from each parent. Children should show corresponding bands to both parents in their fingerprint patterns. This application extends to determining genetic variability within populations - closely related populations show similar fingerprints, while genetically diverse populations display greater variation in their patterns.
Forensic science
Crime scene analysis represents a major application. DNA left at crime scenes (blood, hair, saliva) can be compared with suspect samples. However, matches don't automatically prove guilt - other explanations must be considered:
- Contamination from innocent contact
- DNA from close relatives
- Sample contamination after the crime
Forensic Limitations and Considerations
The probability calculations assume random DNA distribution in the community, which may not apply to certain ethnic or religious groups with limited genetic mixing. This is a critical consideration in forensic applications and legal proceedings.
Medical diagnosis
Genetic fingerprinting helps diagnose diseases like Huntington's disease, caused by repeated AGC sequences on chromosome 4. People with fewer than 30 repeats rarely develop the condition, while those with over 38 repeats almost certainly will. Those with over 50 repeats experience earlier disease onset.
The technique can also identify microbial infections by comparing pathogen fingerprints with known disease organisms.
Plant and animal breeding
Breeding programmes use genetic fingerprinting to prevent undesirable inbreeding and identify individuals carrying specific beneficial alleles. This application includes establishing animal pedigrees and selecting breeding stock to maintain genetic diversity while promoting desired characteristics.
Key Points to Remember:
- Genetic fingerprinting exploits unique VNTR patterns in non-coding DNA sequences to create individual identification profiles
- The five-stage process (extraction, digestion, separation, hybridisation, development) transforms biological samples into distinctive banding patterns
- Gel electrophoresis separates DNA fragments by size, with smaller fragments travelling further than larger ones
- Applications span forensic science, medical diagnosis, paternity testing, and breeding programmes, each requiring careful statistical interpretation
- Results provide probability assessments rather than absolute proof, requiring consideration of alternative explanations in forensic contexts