Manipulating genomes

The genome of an organism refers to all the genetic material it contains.

DNA can be screened to identify potential medical problems.

The human genome consists of 3 million base pairs and 20000 genes.

The principles of DNA sequencing:

Techniques have become faster and more automated than the original Sangar method, but, many of the principles remain the same. Involves terminator bases which stops DNA synthesis.

Terminator bases = A, T, C, G

If terminator base A is used, this will bind where a normal A base would have and instead terminates any further synthesis of DNA.

DNA polymerase, a primer, an excess of nucleotides, terminator bases and the DNA to be sequenced are all mixed together.
The mixture is in a thermocycler for DNA synthesis to occur.
The DNA polymerase will add complementary bases to the DNA, creating a new strand of DNA.
The terminator bases are added at random and will terminate the synthesis of the DNA at different points in each of the replicating DNA strands.
This process continues until all of the possible DNA chains will be produced with the terminator bases added at every possible base.
Each terminator base ( A, T, C, G ) is labelled with a different fluorescent colour so that the end of each DNA fragment can be identified.
The DNA fragments are separated using gel electrophoresis according to their length.
Now each fragment is arranged by length, and the terminator bases are fluorescently labelled a different colour, the sequence of DNA can be worked out.

High through-put sequencing is when many fragments are processed and sequenced simultaneously. This has made the process far more efficient. PYROSEQUENCING.

DNA sequencing = the process of determining the precise order of nucleotides within a DNA molecule.

Genome-wide comparisons:

Genome sequencing has made it possible for scientists to compare the entire genome of individuals of the same species and different species.

Analysing pathogens’ genomes have resulted in many advances, including:

identifying the source of an infection ( e.g. MRSA )
identifying antibiotic-resistant bacteria.
tracking the spread of pathogens to monitor potential epidemics and pandemics.
identifying regions in the genome for new drugs to target.

Comparing genomes has improved the accuracy of the classification of species; our understanding of the evolutionary relationships.

How has gene sequencing allowed for genome-wide comparisons between individuals and between species?…

with technologies such as high throughput pyrosequencing, whole genomes can be sequenced faster.
used to study genotype, phenotype relationships, epidemiology, evolutionary relationships.
the relationship with the genotype and the phenotype is far more different than we thought.

Bioinformatics + computational biology

Synthetic biology:

genetic engineering ( e.g. insulin-producing bacteria )
the use of biological systems in industry ( e.g. immobilised enzymes )
the synthesis of new genes to replace faulty versions of genes ( e.g. replacing the faulty gene that causes cystic fibrosis )
the synthesis of new organisms ( e.g. new bacterial genomes have been created by scientists )

Gene sequencing has allowed for the sequences of amino acids in polypeptides to be predicted. As the mRNA strand can be read as it is apart of the genome. The genetic code specifies the order of the amino acids based on codons.

The principles of DNA profiling and its uses:

DNA profiling is a forensic process used in criminal investigations. Looks at unique patterns in the DNA sequence to identify an individual. Introns contain satellite DNA made up of VNTRs and STRs and they always appear in the same area on chromosomes.

Process: PCR amplifies samples so DNA profiling can be carried out. The fragments are separated by gel electrophoresis according to size/ charge, so, therefore, a DNA profile can be generated for us to see. Different people have different numbers of repeats, so, they have different satellite patterns. DNA profiling helps identify an individual and determine familial relationships.

DNA profiling can also be used to identify people at risk of genetic disorders ( e.g. Huntington’s disease ) before the symptoms appear.

A large DNA sample is required for DNA profiling

Gel lectrophoresis= used to separate DNA fragments based on their size. Can be used to separate proteins, they move through gel during electrophoresis under the influence of an electric current, separated by size and charge.

Benefits and limitations on DNA profiling:

benefits:

can be used in criminal cases to prove innocent or guilty
tiny amounts of DNA can be used
DNA lasts a long time, so, cold cases can be revived by DNA evidence
can be used to find evolutionary relationships

limitations:

can be too dependant on it and ignore other evidence in criminal cases
DNA profiles can be done at different levels and mistakes can be made
contamination if samples with DNA from other organisms

Polymerase Chain Reaction ( PCR ) “Artificial DNA replication”- Amplifies DNA for further study or processing.

Denaturation: 95 degrees Celsius, the high temperatures allow the molecules to gain kinetic energy and vibrate to the point where complementary base pairs separate because hydrogen bonds break. DNA strands separate.
Annealing: thermocycler reduces temperature to 60 degrees Celsius. Annealing of primers which is complementary will bind to the start of the gene you want to amplify. Design primers to recognise target DNA to be amplified.
Synthesis: 72 degrees Celsius. free nucleotides pair up to exposed bases by complementary base pairing. Taq polymerase ( from bacteria ) join up the sugar-phosphate backbone between adjacent nucleotides to form phosphodiester bonds. This will from 2 new strands and the cycle repeats.

Advantages of PCR:

Automated -more efficient
Rapid -100 billion copies of DNA can be made within an hour
Doesn't require living cells- quicker and less complex techniques needed.
used in forensic science with DNA profiling
detects oncogenes, mutations leading to cancer
research- amplifying DNA from extinct organisms
identify viral infections

The principles and uses of electrophoresis for separating nucleic acid fragments or proteins:

Principle- electrical currents are used to separate DNA, RNA or protein fragments depending on their size.

Nucleic acid samples have different rates of mobility when they are different sizes.

The smallest sample of DNA can be collected for genetic fingerprinting, PCR used to amplify DNA.
restriction endonucleases used to break down DNA into fragments.
separate DNA samples using gel electrophoresis. Load into small wells in agar gel which is placed in a buffer liquid with an electrical current.
DNA will move through the gel toward the positive end.
shorter ones move faster and further along the gel.
alkaline added to separate the double strands of DNA.
single strands hybridise ( form hydrogen bonds ) when DNA probes attach
rinse gel and transfer onto nylon sheet, which can b exposed to x-rays to visualise the position of the gene probes or UV light.

The principles of genetic engineering: germ line gene cell therapy

extract desired gene ( e.g. insulin gene ) from human cell and identify using DNA sequencing.
bacteria contains circular DNA and plasmid ( carries extra genes, antibiotic-resistant bacteria, allows for better survival )
restriction endonucleases used to cut genome to extract gene ( produce sticky ends, exposed, recognise plasmid easily ) , then cut with same restriction endonucleases on plasmid, to produce same sticky ends, at blue marker genes (produces blue pigment)
DNA ligase forms phosphodiester bonds between desired gene plasmid to join plasmid and blue marker genes together to form recombinant plasmid, recombinant DNA
place recombinant plasmid in bacteria, make bacterial membrane more permeable, place plasmid inside a vesicle and provide an electric current to fuse them together.
becomes transgenic bacterium
place in nutrient agar plate, culture it, extract bacteria you want and plate with just transgenic bacteria
mass produce insulin

The ethical issues (both positive and negative) relating to the genetic manipulation of animals (including humans), plants and microorganisms:

Pros:

cures for fatal diseases
can create disease-resistant crops
GM crops may be resistant to climate change which will prevent famine as a result of global warming.
if GM seeds are given to poor farmers, it reduces the use of spray pesticides when they plant crops, prevent damage, cheaper

Cons:

animal welfare
transferred genes may mutate
antibiotic genes could spread from marker genes into wild populations and spread antibiotic resistance.

The principles of, and potential for, gene therapy in medicine. To include the differences between somatic cell gene therapy and germ line cell gene therapy:

Replaces faulty alleles which cause genetic disorders.

-Dominant allele disorders= allele is silenced so not expressed.

-Recessive allele disorder= add a functioning dominant allele to be expressed.

Somatic cell gene therapy:

alters body cells, particularly most affected tissues
cells renew so treatment must be repeated regularly
e.g. Cystic Fibrosis treatment, liposomes used to package CFTR alleles, delivered through inhaler, some pass through plasma membrane, if they pass through nuclear envelope, allele can be inserted into the host genome, CFTR protein will be expressed.
functioning alleles are not passed onto offspring
allele may cause a frameshift
allele may be in the wrong location, uncontrollable cell division, cancer risk
short term

Germ line cell gene therapy:

alters the DNA of the gametes or zygotes.
alters cells of the whole organism
changes to the genome
offspring will inherit these changes
illegal in humans
increased risk of cancer
long term