Manipulating genomes — OCR A-Level Biology

DNA sequencing determines the order of nucleotides in a DNA molecule. The classical chain-termination (Sanger) method relies on a template strand, a primer, DNA polymerase and a mixture of normal deoxynucleotides plus modified dideoxynucleotides that terminate chain extension. Because each terminating base carries a distinguishable label, fragments of different lengths are separated by electrophoresis and read in order of size, giving the sequence. Newer techniques, often called next-generation or high-throughput sequencing, avoid gel separation of individual fragments: DNA is fragmented, adaptors are ligated, and millions of clusters are sequenced in parallel, with fluorescent signals detected as each nucleotide is added. These methods generate very large volumes of short reads rapidly and cheaply per base, and the reads are assembled or aligned by computer.

Sequencing whole genomes means the entire DNA content of an organism can be read and compared, not just single genes. Between individuals of the same species, sequencing reveals variation such as single nucleotide polymorphisms and other differences, which can be linked to traits, disease susceptibility or ancestry. Between species, comparing whole genome sequences shows how conserved or divergent genes and non-coding regions are, allowing evolutionary relationships to be inferred: the more similar the sequences, the more recently the species are likely to have shared a common ancestor. Genome-wide comparison also identifies genes shared across many organisms and genes unique to particular lineages. The key idea is that sequencing at genome scale provides the raw data for comparing many genes and regions at once, giving a broader and more reliable picture than comparing isolated genes.

Gene sequencing determines the order of nucleotide bases in a length of DNA. Because the genetic code is a triplet code, non-overlapping and degenerate, each codon of three bases corresponds to one amino acid (or a start/stop signal). Once the base sequence of a gene is known, the reading frame is identified and the codons are translated using a codon table to give the primary sequence of amino acids in the polypeptide. Degeneracy means several codons can specify the same amino acid, so the amino acid sequence is often more reliably predicted than the exact base sequence is from the protein. Comparing predicted sequences between organisms also reveals conserved regions and possible mutations.

Synthetic biology designs and builds new biological parts and systems, or redesigns existing ones. Gene sequencing supplies the raw information: it reveals the base sequences of genes, promoters and regulatory regions, so engineers can identify which DNA elements perform which functions. Sequenced data allow standardised parts to be catalogued, novel gene constructs to be designed and assembled, and the resulting organisms to be checked by re-sequencing. Sequencing also supports comparison across genomes, revealing useful genes that can be transferred into new hosts. In this way sequencing underpins the design-build-test cycle of synthetic biology, from identifying functional DNA elements to verifying engineered constructs.

DNA profiling exploits variation in repetitive, non-coding DNA to produce a pattern characteristic of an individual. In OCR's core method, DNA is extracted and amplified by PCR, cut with restriction enzymes, and the fragments separated by gel electrophoresis; shorter fragments travel further, so band positions reflect fragment lengths. Alternatively, short tandem repeat (STR) numbers at several loci are measured, giving a multi-locus profile. Because close relatives share more bands than unrelated people, profiles are compared rather than read absolutely. Uses include forensic identification from crime-scene samples, paternity and kinship testing, identifying tissue or blood samples, and monitoring genetic diversity in conservation. A match is probabilistic evidence, not proof of guilt.

PCR is an in vitro method that copies a target DNA sequence exponentially. A reaction mixture contains the DNA template, two primers complementary to the flanking sequences, free nucleotides, a thermostable DNA polymerase such as Taq, and buffer. Each cycle has three temperature stages: denaturation near 95 °C separates the strands; annealing around 55–65 °C lets primers bind; extension near 72 °C lets polymerase add nucleotides 5′ to 3′. Repeating cycles doubles the target each time, giving 2ⁿ copies after n cycles. In DNA analysis PCR provides enough DNA for profiling, sequencing or cloning from tiny or degraded samples, and can be made quantitative.

Electrophoresis separates charged molecules by size using an electric field through a gel. Nucleic acids carry negative phosphate groups, so they migrate towards the positive electrode; proteins are separated after denaturation and are usually given a uniform negative charge by SDS, so they too move towards the positive electrode and separate by mass. The gel acts as a molecular sieve: small fragments travel further and faster than large ones in a set time, producing bands. Fragment size is estimated by comparison with a standard ladder of known sizes. Uses include checking PCR products, restriction digests and plasmid digests, separating DNA fragments for sequencing or blotting, and separating proteins for analysis such as Western blotting. The principle is charge-based migration plus size-dependent sieving; the use is resolving and identifying fragments or proteins.

Genetic engineering transfers a gene of interest from one organism into another so the recipient expresses the desired protein. The principles involve obtaining the gene, for example by PCR from DNA or by reverse transcription from mRNA, then inserting it into a vector such as a plasmid. The gene and vector are cut with the same restriction enzyme so the sticky ends are complementary and can be joined by DNA ligase. The vector is introduced into a host cell, often bacteria, and markers such as antibiotic-resistance genes allow transformed cells to be selected. Promoters and, in eukaryotes, appropriate control sequences ensure transcription and translation. The host then expresses the gene, producing the desired protein, which can be harvested.

Genetic engineering means deliberately transferring a gene from one organism into another so the recipient makes a new product. The core techniques form a sequence. A gene of interest is located and cut out using restriction enzymes that recognise specific palindromic sequences and leave sticky ends. A vector, usually a plasmid, is cut with the same enzyme so the ends are complementary. DNA ligase joins gene to vector to form a recombinant plasmid. The vector is introduced into a host cell, often a bacterium, by transformation, for example using calcium ions and heat shock or electroporation. Marker genes such as antibiotic-resistance genes allow transformed cells to be selected. Polymerase chain reaction can amplify the gene, and reverse transcriptase can make complementary DNA from mRNA. Finally the host is cultured and the product harvested.

This criterion asks you to weigh ethical issues, both positive and negative, of genetically manipulating three groups: animals including humans, plants and microorganisms. For animals and humans, positives include treating inherited disease through gene therapy and producing medicines; negatives include welfare concerns in transgenic animals, unforeseen effects of germ-line editing and questions of consent and equity. For plants, positives include improved yield, pest resistance and nutritional content; negatives include gene flow to wild species, reduced biodiversity and corporate control of seed. For microorganisms, positives include producing insulin and vaccines and bioremediation; negatives include escape of modified organisms, horizontal gene transfer and biosafety risk.

Gene therapy treats disease by delivering genetic material into a patient's cells so a faulty gene is corrected, replaced or supplemented, or so a harmful gene product is suppressed. Somatic therapy targets body cells such as lung epithelium or bone marrow; changes are not inherited. Germ-line therapy would alter gametes or embryos and is not used clinically in humans. Delivery may be in vivo (vector introduced directly, e.g. an adenovirus carrying a functional allele to lung cells) or ex vivo (cells removed, modified, checked and returned). Vectors include modified viruses and liposomes. Success depends on efficient, targeted delivery, stable expression and immune tolerance. Potential is greatest for single-gene recessive disorders such as cystic fibrosis or severe combined immunodeficiency; complex polygenic disease is far harder.