DNA Sequencing and Profiling (OCR A-Level Biology A): Revision Notes

DNA Sequencing and Profiling

DNA sequencing techniques

DNA sequencing determines the specific order of nucleotide bases (adenine, thymine, cytosine, and guanine) along a DNA molecule. The sequence information reveals the genetic instructions encoded within genes and provides insights into evolutionary relationships, genetic variation, and disease mechanisms.

Chain-termination method

The chain-termination method, also known as Sanger sequencing, was developed in the 1970s and represented a breakthrough in molecular biology. This technique works by copying short fragments of single-stranded DNA template, similar to the polymerase chain reaction (PCR).

The key innovation involves adding modified nucleotides called dideoxynucleotides to the reaction mixture. These modified nucleotides lack the 3' hydroxyl group needed to form the next phosphodiester bond, so when incorporated, they terminate DNA synthesis at that position. By including different dideoxynucleotides for each of the four bases (A, T, C, G), the reaction produces multiple DNA copies of varying lengths, each ending at a specific base position.

Approaches to genome sequencing

Two main strategies emerged for sequencing complete genomes:

Traditional genetic mapping began by identifying the chromosomal locations of genes using data from genetic crosses and pedigree analysis of families affected by genetic diseases such as Huntington's disease and haemophilia. This approach provided a framework for understanding genome organization.

Shotgun sequencing offered a complementary approach by sequencing random DNA fragments without prior knowledge of their genomic location. After sequencing many fragments, bioinformatics tools compared the sequences to identify overlapping regions, allowing the fragments to be assembled like pieces in a very long, linear jigsaw puzzle. This method was first successfully applied to sequence the nearly $2$ million base pair circular chromosome of the bacterium Haemophilus influenzae. By 2001, combining shotgun sequencing with traditional mapping approaches produced the first draft of the human genome sequence.

Next-generation sequencing

The chain-termination method has now been superseded by next-generation sequencing (NGS) technologies. These advanced methods can sequence thousands to millions of DNA molecules simultaneously without requiring separate electrophoresis steps. Different detection systems identify the bases as they are incorporated during DNA synthesis.

One example is pyrosequencing, which uses the enzyme luciferase to emit flashes of light when pyrophosphate (P-P) is released during nucleotide addition to the growing DNA strand. Each flash indicates that a specific base has been incorporated, allowing the sequence to be read in real time.

Timeline of DNA sequencing developments

Year	Development
1975	Chain-termination method developed by Fred Sanger and Andrew Coulson in the UK; chemical degradation method by Alan Maxam and Walter Gilbert in the USA
1977	First complete genome sequenced: bacteriophage φX174 with $5386$ nucleotides
1990s	Development of capillary flow electrophoresis with appropriate DNA detection systems; shotgun approach used for Haemophilus influenzae genome
1996	Yeast (Saccharomyces cerevisiae) genome sequenced – first eukaryote completed
1998	Sequencing machines with $96$ capillary sequencers developed, termed high-throughput sequencing
2001	First draft of human genome published (complete version in 2004)
2005	First next-generation sequencing machine became commercially available
2012	Tomato (Solanum lycopersicum) genome published using both chain-termination and next-generation methods – probably the last genome sequenced using Sanger sequencing
2015	Genome of extinct woolly mammoth sequenced

Nanopore sequencing

An emerging technology promises to revolutionize DNA sequencing by making it portable and even more accessible. Nanopore sequencing works by threading a DNA molecule through a tiny protein pore embedded in a lipid bilayer membrane. As the DNA passes through the pore, ions flow through the channel, creating an electrical current. Each of the four bases (A, T, C, G) alters the current flow in a distinctive way, allowing the sequence to be determined in real time.

Applications of whole-genome sequencing

The reduced costs of next-generation sequencing have enabled genomes from numerous species to be sequenced. Comparing whole genomes allows scientists to locate the same or similar genes across different species and analyze evolutionary relationships.

Sequencing genomes from many individuals within the same species reveals information about genetic variation within populations. This has been applied to humans and other mammals, particularly domesticated animals (horse, cattle, pig, yak) and endangered species such as giant pandas. Important crop plants including rice, papaya, cabbage, potato, wheat, and tomato have also had their genomes sequenced.

Synthetic biology

Understanding genome sequences enables researchers to manipulate genetic systems in novel ways. Synthetic biology is a new science that involves designing new biological systems and redesigning existing systems using knowledge of nucleic acids and cell biology.

Scientists have used genome information to perform "knockout" experiments, systematically removing genes from simple organisms like Mycoplasma to determine the minimum number required for life. Similar studies in other prokaryotes and in yeast have revealed essential gene sets.

This capability allows for the production of novel molecules, such as enzymes for medicinal drug production. Existing genes can be modified to improve protein function, as demonstrated by the various forms of human insulin now available for diabetes treatment. Entire cellular systems can be redesigned to make genetically modified organisms work more efficiently in producing desired proteins.

Epidemiology applications

Whole-genome sequencing of pathogens has transformed epidemiology – the study of disease spread in populations. Sequencing enables epidemiologists to identify specific strains infecting people, livestock, and crops, determining the most appropriate control methods. For example, genome sequencing of the Ebola virus contributed significantly to understanding disease transmission during the 2015 West African epidemic, helping to track the spread and implement targeted interventions.

DNA profiling

Genome sequencing has revealed extensive variation in DNA between individuals within a species. This variation makes it possible to identify individuals with very high accuracy, since the probability of two individuals having identical DNA is extremely small (unless they are clones).

Repeated sequences in DNA

A distinctive feature of genomes is the presence of nucleotide sequences that are repeated, often many times. These repeated regions can be treated as genetic markers, with the variable number of repeats functioning like different alleles. Because these regions show high variability between individuals, they represent examples of genetic polymorphism.

Before high-throughput sequencing became available, individual identification relied on detecting differences in phenotypes, such as using protein electrophoresis to identify sickle cell anaemia carriers or using antibodies to determine blood groups. The discovery of repeated DNA sequences led to methods of genetic profiling (often called genetic fingerprinting).

Short tandem repeats

The use of minisatellites has been largely replaced by short tandem repeats (STRs), also known as microsatellites. These are much shorter than minisatellites, consisting of $2$–$5$ nucleotides repeated $10$–$30$ times. For instance, the sequence CACACA might be repeated between five and twenty times at a particular genomic location.

The number of repeats at any STR locus varies between individuals. This variability arises from the inaccuracy of DNA polymerase when copying these repetitive regions during replication. Although the error rate is generally very low, occasional mistakes lead to changes in the number of repeat units, creating new alleles.

STR markers can be classified as:

• Simple: identical-length repeats
• Compound: two or more adjacent repeats
• Complex: several different length repeats

STRs occur on all $22$ autosomal chromosomes as well as both X and Y sex chromosomes. The CAG repeat sequence in the huntingtin gene represents one example of an STR. In DNA profiling, the alleles at selected STR loci are determined. Many of the most useful STRs for identification purposes are located in non-coding DNA between structural genes.

Forensic DNA profiling

Forensic scientists use DNA profiling to identify crime victims, suspects in criminal cases, disaster victims, and missing people. The European DNA 17 profile consists of $16$ STR loci plus markers that identify sex, providing a highly discriminating identification system.

Characteristics of forensic STR markers

STRs selected for forensic applications typically have four- or five-nucleotide repeats. These were chosen because they:

• Show high degrees of polymorphism, providing excellent discrimination between individuals
• Are robust and suffer only low levels of environmental degradation
• Provide high-quality, error-free data
• Represent discrete alleles that are clearly distinguishable from one another
• Require only small sample amounts (as little as $100$ picograms from about $16$ cells)
• Are located in non-coding DNA, so no selective pressure acts against any alleles
• Can be efficiently amplified by PCR, including multiplex PCR of multiple loci simultaneously
• Produce low levels of artefact formation during amplification

STR analysis process

PCR amplifies the DNA samples to produce many copies of the regions containing the STRs. The STRs are labelled with fluorescent dyes, and the sequences are separated by gel electrophoresis or detected during PCR using four different fluorescent colours (typically red, green, yellow, and blue). A laser scanner detects the fluorescence as each fragment passes the detector.

DNA profiling and disease risk

Genetic testing

DNA testing or genetic testing detects the risk of genetic diseases or disorders. DNA samples typically come from blood samples or buccal swabs (cotton swabs wiped inside the cheek to collect epithelial cells). Almost all molecular genetic tests use PCR to amplify specific DNA regions.

Because PCR is highly selective, it can amplify just the DNA section needed for testing. However, PCR typically amplifies only short sequences (up to about $2000$ bases). Since most genes are larger than this, multiplex PCR reactions are needed to copy entire genes.

Genetic tests exist for many monogenic diseases, including:

• Cystic fibrosis
• Huntington's disease
• Sickle cell anaemia
• β-thalassaemia
• Haemophilia A and B
• Severe combined immunodeficiency syndrome
• Some forms of breast cancer

These diseases result from inherited mutations that run in families. When someone tests positive for a disease-causing mutation, genetic tests are often offered to other family members. Some people know a particular disease runs in their family and choose genetic testing. However, in some cases (such as haemophilia), up to 40% of affected individuals come from families with no previous disease history.

Single nucleotide polymorphisms

Single nucleotide substitutions cause most genetic diseases. Single nucleotide polymorphisms (SNPs), pronounced "snips", are the simplest and most common type of genetic variation, comprising around 90% of genetic variation in humans. They occur during meiosis when DNA is replicated.

A SNP represents a change of a single base at a specific position in the DNA sequence. For example, a cytosine might be replaced with a thymine in a particular DNA stretch. This does not change the DNA length, so if the SNP falls in an exon, there is no effect on the gene's reading frame during protein synthesis.

Different human genome sequencing projects have revealed that individuals have between $3$ and $4$ million SNPs compared to the reference sequence. So far, over $30$ million SNPs have been discovered from human genome sequencing projects.

Personalised medicine

The availability of inexpensive next-generation sequencing techniques, such as nanopore sequencing, will allow doctors to determine the sequence of specific genome regions. This information enables the prescription of drugs that will be effective for people with particular genotypes. This approach represents personalised medicine, where treatment is tailored to each individual's genetic makeup, potentially improving outcomes and reducing adverse drug reactions.