Understanding and manipulating DNA
Discovery of the structure of DNA in 1953. There has been a number of advances in science that have used this knowledge.
- DNA PROFILING-Genetic fingerprinting-used in forensic crime scene analysis and paternity and maternity testing.
- Genomic sequencing and comparative genome mapping, used in research into function of genes and regulatory DNA sequences.
- GENETIC ENGINEERING-used in the production of pharmaceutical chemicals, genetically modified organisms and xenotransplantation.
- GENE THERAPY-used to treat conditions such as cystic fibrosis
Gene technology is advancing rapidly, but it is also important to note that many of the techniques involved have their basis in natural processes.
- DNA strands can be cut up into smaller fragments using restriction endonuclease enzymes.
- The fragments can be separated by size using electrophoresis and replicated many times to produce multiple copies using the polymerase chain reaction.
- DNA fragments can be analysed to give their specific base sequence
- DNA fragments can be sealed together using ligase enzyme
- DNA probes can be used to locate specific sequences on DNA fragments
Such techniques mean that sections of DNA, including whole genes, can be identified and manipulated.
The Genomic Age
DNA contains genes which code for the production of polypeptides and proteins.
However, this coding DNA forms only a small part of the DNA found in an organism.
Only 1.5% of the genome of humans actually codes directly for polypeptides and proteins.
Much DNA is non-coding DNA and has been referred to as junk DNA.
'Junk' is misleading as this non-coding DNA carried out a number of regulatory functions, many of which are still to be discovered.
A great deal of research is going on into trying to work out how genomes work as a whole.
Genomics-the study of genomes-is seeking to map the whole genome of an increasing number of organims.
Comparing genes and regulatory sequences of different organisms will help us to understand the roe of genetic information in a range of areas including health, behaviour and evolutionary relationships between organisms.
Sequencing the genome of an organism
The sequencing reaction can only operate on a length of DNA of about 750 base pairs. This means the genome must be broken up and sequened in sections. In order to ensure that the assembled code is accurate, sequencing is carried out a number of times on overlapping fragments, with the overlapping regions analysed and put back together to form the completed code.
- Genomes are first mapped to identify which part of the genome they have come from. Information that is already known is used-using the location of microsatellites-short repetitive sequences of around 3-4 bases at many locations on the genome.
- Samples of the genome are sheared into smaller sections of around 100,000 base pairs. Sometimes called the 'shotgun' approach.
- These sections are places into separate bacterial aritificial chromosomes-BACs and transferred to E.coli cells. As the cells grow in culture, many copies of the sections are produced. These cells are referred to as clone libraries.
Sequencing the BAC section
- Cells containing specific BACs are taken and cultured. The DNA is extracted from the cells and restriction enzymes are used to cut it into smaller fragmets. The use of different restriction enzymes on a number of sample gives different fragment types.
- The fragments are separated using a process known as electrophoresis.
- Each fragment is sequenced using an automated process.
- Computer programmes then compare overlapping regions from the cuts made by different restriction enzymes in order to reassemble he whole BAC segment sequence
A wide variety of organism genomes have now been sequenced. Knowing the sequence of bases in a gene of one organism and being able to compare genes for the same proteins across a range of organisms is known as comparative gene mapping. This has a wide ranfe of applications;
- The identification of genes for proteins foung in all or many living organisms gives clues to the relative importance of such genes to life
- Comparing the DNA/genes of different species shows evolutionary relationships. The more DNA sequences organisms share, the more closely related they are likely to be.
- Modelling the effects of changes to DNA/genes can be carried out. e.g. a number of studies have tested the effects of gene mutations on genes obtained from yeast that are also found in the human genome. Yeast is a haplois organism, so a mutation to a gene is always shown in the phenotype
- Comparing genomes from pathogenic and similar but non-pathogenic organisms can be used to identify the genes or base-pair sequences that are most important in causing the disease. This can lead to identification of targets for developing more effective drug treatments and vaccines.
- The DNA of individuals can be analysed. This analysis can reveal mutant alleles, or the presence of alleles associated with increased risk of disease, e.g. heart disease or cancer