Manipulating genomes (6.1.3)
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DNA sequencing can be used to determine the sequence of base pairs in an organism’s genome.
The Human Genome Project successfully sequenced the first complete human genome. Since then, thousands of human genomes have been sequenced.
Only about of the human genome is made up of coding DNA, which directly codes for proteins. The remaining 98 of the human genome is non-coding DNA, which does not directly code for proteins, but has other useful functions.
The capillary method is one of the earlier methods for DNA sequencing:
- The DNA is chopped up into fragments using enzymes.
- DNA primers, DNA polymerase, terminator bases and an excess of free nucleotides are added to the fragments. The terminator bases stop replication when they are included in a sequence.
- PCR is carried out, replicating the DNA fragments.
- The strands are separated by capillary sequencing, which is similar to electrophoresis but performed in capillary tubes.
- The terminator bases are used to identify the final base in the sequence.
The data is then compiled into a mapped genome. Historically, this was a manual process.
Over time, sequencing techniques have been developed, such as high–throughput sequencing. This process involves using a slide called a flow cell, in which fragments can be replicated in situ by PCR and sequenced simultaneously.
This process requires less time to map the fragments because it can be carried out by a computer. The software analyses the fragments and collates the information into the mapped genome.
As DNA sequencing has become cheaper and quicker the range of uses has expanded. Now human genomes are analysed for inheritance patterns and many health conditions, such as Alzheimer’s, have been associated with genetic markers.
Epidemiologists study human health at a population level. DNA sequencing allows epidemiologists to examine genetic and lifestyle factors together and assess how diseases develop and are inherited.
DNA sequencing can aid in identifying species.
It can be challenging to determine whether two organisms are different species purely using observation. DNA sequencing can provide additional information to help determine whether an organism is a different species.
DNA sequencing can also be used to determine the evolutionary relationship between two organisms, build evolutionary trees and identify common ancestors. This can be used to determine how closely or distantly related two organisms are.
The proteome consists of all the proteins produced by an organism’s genome.
Pre-mRNA contains introns and exons. Each gene can code for multiple different proteins when the pre-mRNA is spliced in different ways prior to translation.
Proteomics can aid in understanding how genes code for multiple proteins, depending on how the pre-mRNA is altered.
Synthetic biology involves redesigning natural biological systems or artificial biological pathways.
Synthetic biology can include:
- Genetic engineering.
- Using biological systems in industry, such as making insulin.
- Gene therapy, such as replacing faulty genes which cause diseases.
- Synthesising new organisms such as bacteria.
DNA profiling (also called DNA fingerprinting) analyses non-coding, variable regions of DNA to compare individuals.
DNA profiling most commonly uses short tandem repeats (STRs), also known as microsatellites, which consist of short repeated DNA sequences. The number of repeats at each STR locus varies between individuals, producing a unique DNA profile (except in identical twins).
DNA profiling follows these steps:
- Sample collection – a biological sample containing DNA is obtained, such as blood, saliva, hair roots or plant tissue.
- DNA extraction – DNA is isolated from the cells in the sample.
- PCR – the short tandem repeat regions (STR) are amplified using PCR to produce many copies for analysis.
- Electrophoresis – the amplified DNA fragments are separated according to their length (smaller fragments move further), producing a pattern of bands.
- Profile comparison – resulting DNA profiles are compared between samples. Matching banding patterns indicate that the DNA is likely to have originated from the same individual.
Relatives share a predictable proportion of STR patterns:
- Parents and offspring share approximately of their STR alleles / bands.
- Siblings share many STR alleles / bands, but usually have different overall DNA profiles.
DNA profiling can be used to confirm parentage, breeding lineages, or inheritance disputes. In plants, profiling is used to confirm breeding crosses and verify cultivar purity. In animals, it is used to manage captive breeding programmes (zoos) and maintain genetic diversity.
Uses of DNA profiling include:
- Matching suspects to crime scene samples.
- Identifying victims in disasters.
- Distinguishing between similar-looking organisms (rare species, plant cultivars).
- Preventing inbreeding by checking relatedness.
- Tracking illegal wildlife trade (timber, ivory).
- Assessing the genetic risk of disease.
DNA fingerprinting can be used for determining genetic relationships.
All the bands shown in a child’s DNA fingerprint must correspond to bands from one of the parents’ fingerprints. It is important to note that not all the bands from either parent will be observed in the child.
PCR (Polymerase Chain Reaction) is a technique used to rapidly amplify specific fragments of DNA.
The reaction mixture contains:
- Template DNA – the DNA containing the target sequence to be copied.
- Primers – short single-stranded DNA sequences that bind to the ends of the target region.
- Free DNA nucleotides – used to build new DNA strands.
- DNA polymerase – a heat-stable enzyme that synthesises new DNA strands.
- Buffer – maintains the optimum and conditions for the enzyme.
The PCR Cycle (repeated 25–35×):
- Denaturation (~95): hydrogen bonds between complementary DNA strands break.
- Annealing (~50–65): primers bind (anneal) to complementary sequences on the template (target) DNA.
- Extension (~72): DNA polymerase adds complementary nucleotides to the primers creating new DNA strands.
Each cycle approximately doubles the amount of target DNA resulting in exponential amplification.
After n cycles, the number of DNA molecules ≈ 2n (assuming 100 efficiency).
PCR–based DNA replication has many advantages. PCR:
- Can replicate DNA extremely quickly.
- Does not require any living organisms or cells, so has no ethical issues.
- Can be used for a variety of applications, such as forensics.
The main disadvantage of PCR is that if the sample is contaminated, then the contaminated DNA will also be replicated.
Once DNA has been amplified and replicated using PCR, electrophoresis can be used to separate DNA. Electrophoresis can also be used to separate proteins.
The charged particles move through a gel medium as a current is applied.
DNA is negatively charged due to the phosphate groups, so it will move towards the positive electrode (anode).
The fragments will be separated by size as the smaller fragments move faster through the gel.
A ladder is also used, which contains DNA fragments of known lengths. This acts as a calibration and can be used to compare DNA fragments and estimate their lengths.
Genetic engineering involves manipulating an organism’s genome. One of the most common ways this is carried out involves inserting desired genes into a bacterial plasmid, creating recombinant DNA.
This recombinant DNA can be inserted into an organism, creating a transgenic or genetically modified organism.
Gene insertion is possible as transcription and translation are universal biological processes.
Enzymes called restriction endonucleases can be used to cut the desired gene from an organism.
Restriction endonucleases can cut a gene in different ways, producing either blunt ends or sticky ends.
The mRNA of the desired gene for genetic engineering can also be isolated from the main strand. This method is easier when the desired gene is being isolated from an organism with a large genome.
The mRNA for the desired gene is isolated, then treated with reverse transcriptase enzymes. This produces complementary DNA (cDNA).
Following extraction, the desired gene is placed into a vector, which is usually a bacterial plasmid.
The plasmid is edited to contain two markers: one is a selectable marker, such as a gene conferring antibiotic resistance, and the other is a detectable marker, such as a fluorescent marker or an enzyme that changes colour under specific conditions.
The plasmid is opened at the same site as the marker genes using a restriction endonuclease. DNA ligase joins the plasmid and the desired gene together. This results in one of the marker genes failing to function.
The functional markers allow screening to determine which bacteria have taken up the desired gene.
Once the recombinant vector has been produced, it must be transferred into the target organism.
A calcium-rich solution can be used to make the bacterial cell membrane porous and able to take up the plasmid.
Electroporation is when an electric current is used to make the cell membrane more porous. Electroporation can be used for both bacterial and eukaryotic cells.
Following the take-up of the successfully recombinant plasmid shown above, the host bacteria will exhibit antibiotic resistance but will not fluoresce. The bacteria are then allowed to multiply on an agar plate infused with antibiotics. This allows only the correct bacteria, the non-fluorescent spots, to be extracted and grown to create the cell culture.
Electrofusion can be used to create genetically modified organisms.
A small electric current can be applied to cell membranes. This causes cells to fuse, forming polyploid cells: cells with more than two paired chromosomes.
Electrofusion works best with plant cells as polyploid animal cells do not survive inside of an organism for long.
Electrofusion can be used to create monoclonal antibodies.
Animals can be genetically modified to produce drugs and proteins, this is called pharming.
Mammals, such as sheep and goats, can be genetically modified so that proteins, such as proteins involved in clotting, are produced in their milk. These proteins can then be purified and used medicinally to treat conditions such as haemophilia.
Genetically modified plants can be produced using the bacteria Agrobacterium tumefaciens. This bacterium causes tumour growth in plants. Rapid mitosis in tumours can be utilised to transfer desired genes into plant cells.
Plants can be modified to be resistant to weed killers, such as glyphosate, or to produce their own insecticides.
Gene therapy involves treating genetic diseases which are caused by faulty alleles, such as haemophilia, cystic fibrosis and sickle cell disease.
These diseases may be treated by replacing the faulty allele with a functioning allele.
Somatic gene therapy inserts the functioning allele into the affected organisms’ somatic cells. This has been used to treat genetic disorders and also certain types of cancer. Somatic cells are diploid cells and do not include sperm or egg cells.
This is only a temporary solution, and repeat treatments would be required. As the modified cells have short natural lifespans, they are eventually replaced by stem cells, and the functional allele would no longer be present.
Germ line cell gene therapy inserts the functioning allele into a gamete or the embryo after fertilisation.
This is a permanent treatment, as the functional allele would be expressed in the stem cells and be passed to all the organism’s cells as the cells in the embryo replicate.
The corrections from germ line gene therapy are passed to future offspring.
