Manipulating Genomes - module 6.3 Biology OCR A, notes PDF

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Manipulating Genomes

PCR Polymerase Chain Reaction. Method of amplifying DNA by artificial replication in vitro (In glass, outside body) Useful as it allows a large quantity of DNA to be produced from a small sample. Required Components: • Original DNA sample that is being replicated. Can only be a short sequence of around 10000 base pairs • Nucleotides (that are free in solution) A, T, C and G. These are the building blocks used to synthesise the new DNA chain. • DNA polymerase in the form of Taq polymerase. It is stable at high temperatures which allows for a fast rate of reaction. • Small primer sequences of DNA which are complementary to 3’ end of the DNA sample. It proves a small double-stranded region of DNA for Taq polymerase to bind to. • A thermocycler- a computer-controlled machine which controls temperature for a number of cycles. It is automated which makes the process faster. Stages: One- Denaturation Two- Annealing Three- DNA synthesis

Mechanism: The DNA sample, nucleotides, Taq polymerase and smaller primer sequence are mixed in PCR tube

Temperature is raised to 72 which is the optimum temperature for Taq polymerase

PCR tube is placed into thermocycler.

Temperature is decreased to 55-68, allowing primer sequence to bind to the 3’ ends of the DNA strands

Taq polymerase binds to the double stranded ends of DNA and synthesises two new complementary strands of DNA

Temperature is increased to 95C which breaks the hydrogen bonds between the two strands of sample DNA

Sample DNA separates into two single strands

This results in the synthesis of two new double stranded molecules that are identical to the original sample

After one cycle, two molecules of the double stranded sample DNA are formed. This means the amount of DNA produced after each cycle is 2 to the power of the number of cycles. Therefore the amount of DNA increases exponentially each cycle. As each cycle only takes 2 minutes, vast amplification will only take a few hours. Applications of PCR The amplification of DNA using PCR allows for DNA to analysed using other techniques such as DNA sequencing, profiling and gel electrophoresis. The vast quantity of DNA produced allows for replication. Forensic application: - Small samples of DNA that are obtained from evidence left at crime scenes like blood, skin cells or hair can be amplified using PCR and then used for DNA profiling. Results can be matched against possible suspects. - DNA profiling can also be used in establishing parentage for immigration or child custody cases. Medical application: - PCR used to gather enough DNA so it can be used for DNA sequencing which allows scientists to detect mutations. This is useful in cancer patients where they can determine the type of mutation and then tailor treatment to the individual. - Also used for tissue typing to see whether tissue from a donor is compatible with the recipient, reducing risk of rejection. - Also can be used to detect small traces of viral DNA in cells or blood streams. This can be used to detect viruses such as HIV quite early on so antivirals and other treatments can start earlier. This is quicker than detecting viruses via Antibodies. - PCR can also be used to monitor spread of diseases in a population, allowing detection of new pathogens and preparation for outbreaks. Research application: - PCR can be used to amplify DNA samples of ancient species which can be used to determine evolutionary relations - Also used for research into gene expression in cells, allowing for understanding of how genes work.

DNA Profiling/Genetic Fingerprinting It is a method to produce a specific pattern of DNA bands from an individual’s genome. Every individual has a unique genome and therefore a unique DNA profile, except monozygotic twins. It relies on short, repeating sequences o fDNA that are found in the non coding region. The repeating sequences are variable number tandem repeats (VNTRs). They are found in more than 1000 different locations in the hum genome. VNTRs at each loci will differ in number of repeats, so every individual has VNTRs of different lengths which results in the unique DNA profiles. DNA is extracted from tissue samples such as: cheek cells from mouth swabs, blood, hair or skin cell remains and accent DNA from bones

- The DNA is amplified using PCR - It is digested by cutting it into fragments using restriction endonuclease -

which cut up DNA at recognition sites- specific sequences of bases. They leave the VNTRs intact AS VNTRs differ in individuals, the fragments will differ in size The DNA fragments are separated out using gel electrophoresis. The smaller fragments travel quicker and further along the gel whereas the larger fragments move slower and move less far along the gel. Gel produces a banding pattern that is unique to each individual The banding pattern can be visualised using dyes or probs that are radioactive or fluorescent The banding pattern is the DNA profile.

Once each DNA profile is produced, they can be compared. If two samples are the same it suggests that they were taken from the same person. If the DNA profiles from two different samples share around 50% DNA bands it suggests there is a genetic relationship. Applications of DNA profiling: • Forensic science analysis for crime • Determining genetic relationship, eg for paternity disputes or immigration cases • Identifying individuals at risk of a disease- some VNTRs are associated with certain diseases. For example the number of CAG repeats and Huntington’s disease.

Electrophoresis Electrophoresis is the process of separating DNA fragments (or other macromolecules) by their size via an electric current. Gel electrophoresis requires: 1. DNA molecule to be separated into smaller fragments 2. An agarose gel plate which is the medium. It contains hollow wells at one end in which the DNA fragments are pipetted into 3. Electrophoresis tank- holds gel plate and has electrodes connected to power supply. This allows a current to pass between a cathode and an anode. 4. Buffer solution- ion containing solution which immerses the gel plate 5. DNA loading dye- allows scientists to track how far DNA has moved 6. DNA ladder. Contains fragments of DNA of known length which the sample of DNA can be compared to. Process: - DNA loading dye is added to the PCR tubes containing the different DNA samples. - Different DNA samples are pipetted into the wells - First well is filled with DNA ladder - An electric current is applied across the gel plate and the gel is said to. be ‘running’ - The DNA moves towards the positive anode, this is because it contains phosphate groups which makes it have an overall negative charge - Smaller fragments of the DNA move faster and therefore travel further - The gel is usually left running for a few hours, however this can be sped up by increasing voltage Visualisation: - DNA loading gel allows DNA in the gel to be tracked but does not provide high quality visualisation. - DNA binding gel is added to the gel which is radioactive or fluorescent, allowing DNA bands to be seen under X-ray or UV light. - DNA probes are shorter sections of DNA that are complementary to a known DNA sequence. They can be fluorescent or radioactive. In order to use them, DNA must be made single stranded which is done by immersing it in alkaline solution. Nitrocellulose paper or nylon membrane is placed over the gel and absorbs alkaline and transfer single stranded DNA to paper in the same position it was located on the gel. The probs are

added which bind to specific fragments of DNA by complementary base paring by hybridisation. Then seen via UV light or X-ray. Protein Gel Electrophoresis Unlike DNA Proteins have different surface charges which affects their speed as they move through the gel. Therefore charged detergents must be added before running the gel to give them a uniform negative charge. This allows proteins to be separated according by molecular mass. This is used to diagnose genetic conditions caused by mutant proteins.

DNA Sequencing DNA sequencing is the process used to determine the prise order of nucleotides in a length of DNA. Radioactive Sanger Sequencing Process: • DNA to be sequenced is placed in four separate tubes and is mixed with: primers, activated free nucleotide bases, DNA polymerase and Radioactive marked chain terminating nucleotides. • The tubes are placed in a thermocycler and PCR begins. • The DNA is heated up until it denatures into single strands • Temperature is lowered and primer anneal to one of the strands which is used as a template for sequencing • DNA polymerase is able to bind so DNA synthesis begins • The chain terminating nucleotides are randomly incorporated, stopping synthesis • The PCR cycle is repeated and another chain terminating nucleotide is added • The many cycles of PCR results in many fragments being formed that differ in length by one base • The DNA fragments are then pipetted into well of arose gel for gel electrophoresis, which senates fragments by length as the shorter fragments move further than the longer fragments • The bands of DNA can be visualised as the radioactive chain terminating nucleotides expose photographic paper to create an autoradiogram. Analysis: To work out DNA sequence, bands are read off the autoradiogram starting from the bottom. Process is manual so is time consuming. Fluorescent Sanger Sequencing • • • •

Carried out in an automated sequencing machine. Uses fluorescent markers on chain terminating nucleotides. Only requires one PCR reaction per DNA fragment as each has own colour Mixture used in PCR contains: Many fragments of DNA, DNA polymerase, primers, activated free nucleotides and low proportion of coloured chain terminating nucleotides.

• Results in many fragments of DNA that differ by one base • Fragments are separated according to length via capillary gel electrophoresis. The smaller fragments move faster through the capillary tube. • At the end of the fragment the laser detects the fluorescent marker • DNA recorded by chromatogram with is used to read the sequence of the bases. Next Generation sequencing - New techniques of DNA sequencing - They are all massively parallel meaning many DNA fragments can be sequenced at the same time - They have a high throughput which means a large amount of sequence data can be generated from one sequencing reaction - They are fast as DNA fragments are sequenced at the same time as DNA synthesis - They are cheap as more sequence data is generated per reaction Pyrosequencing Detects presence and intensity of light. Setup: • DNA molecule is broken up into many fragments that are around 300 to 800 bases in length • Fragments are separated into single strands and one strand is fixed to the flow cell (a plastic slide) which contains wells with a strand of DNA each. The single strands act as a template. • Flow cell is incubated in reaction mixture containing: primer, enzymes (DNA polymerase, ATP sulfurylase, Luciferase and apyrase), APS and Luciferin Process: • First reaction takes place by adding activated nucleotides that contains one of the four bases of the flow cell • If base of the template is complementary to the base added, the nucleotide will be incorporated to the DNA chain by DNA polymerase • This results in a release of diphosphate/pyrophosphate • The pyrophosphate reacts with the APS to form ATP which is catalysed by ATP sulfurylase • Luciferase uses the ATP to catalyse Luciferin to oxyluciferin, generating visible light

• Any nucleotides that weren’t incorporated into the DNA are degraded by apyrase • A different activated nucleotide is washed across the flow cell, resulting in another flash of light if incorporated • Cycle repeats until DNA fragments have been fully synthesised. Determining DNA sequence: • When a nucleotide is incorporated into a strand of DNA the flash of light occurs in its individual well of the flow cell • When multi nucleotides get incorporated the flash of light is more intense as more ATP is synthesised so more luciferin is being converted to oxyluciferin. • A camera detects difference in light intensity between one and three nucleotides being incorporated • If well does not flash with light, nucleotide will not be incorporated into that DNA strand • The sequence of DNA strand can be read from the sequences of flashes from each well when each nucleotide is washed over flow cell. Whole genome sequencing Sanger and next generation sequencing methods allow short lengths of DNA to be sequenced. Genomes contain a vast number of base pairs. In order to work out whole genomes scientists sequence many small overlapping regions of DNA. A computer analyses the sequences and works out how to reassemble the genome from he areas of overlap. This process used to be time consuming and expensive, however this has improved as technology has developed. One new project for whole genome sequencing is the 100k genome project which looks to sequence 100,000 genomes from NHS patients and families to see how genomes link to certain diseases. The branch of biology which stores and analyses genome data is bioinformatics. Bioinformatics can be used to: - Develop algorithms to assemble whole genomes from many small overlapping sequences of DNA - Compare genomes across individuals of one species to find links between genes and disease - Compare whole genomes across species to work out evolutionary relationships

- Predict primary structure of proteins from gene sequences. Comparing Genomes Sequencing whole genomes allows many genomes to be compared to each other. Between Species Very few genes are unique to a particular species. Differences between organisms are not always due to having different genes. It can be due to mutations within shared genes or different in regulation. Phylogeny: - Genome sequencing allows for determining evolutionary relationships - Genomes of closely related species can be compared to determine genetic similarity. - Mutations occur at a basic rate so can be calculated - The genetic similarity can be used to determine how long ago the species diverged. - The more similar the genome the more recently their common ancestor lived. Classification: Genome sequencing has resulted in reclassification of organisms as before hand they were classified based on how similar they looked morphologically, however genome comparison is more accurate. Between Individuals Comparing genomes has been used in medicine. From the 10,000s of human genomes that have been sequenced, scientists have estimated around 0.1% of our genomes differ. This is around 3 million nucleotide bases and it is due to mutations that occur at SNPs. Certain SNPs determine our suspect ability to a wide range of diseases. These can be genetically tested for which allows identification of possible medical diseases and early intervention. Most non-communicable diseases are not caused by just one SNP, many genes interact with each other or the environment affecting likelihood of developing diseases.

Sequencing bacterial genomes allows doctors to determine whether strains are resistant to certain antibiotics, allowing for effective treatment. Comparison of bacterial genomes allows doctors to determine the source and progress of disease outbreak. Comparison of genomes of pathogens from different outbreaks of the same disease can reduce the chance of a further outbreak. Genomes to Proteomes A proteome is all the proteins produced by the genome. This means the genome determines the proteome, which can be worked out by the DNA sequence of every gene. The genetic code determines the primary structure of the protein. Computational biology can be used to predict the tertiary structure and potential functions of proteins. Simple organisms: - Cheap and easy. - Prokaryote genomes are shorter than eukaryotic genomes and are not associated with histone proteins, so are easier to sequence. They also do not have non coding regions of DNA. - Single celled Eukaryotes are easier to sequence than multicellular organisms as they have fewer genes - Determining protein of pathogens is useful as it can help identify proteins of the cell surface that may act as antigens. This can be sed to develop vaccines. Multicellular Organisms Difficult and complicated because: • Only 1.5% of DNA in human genomes code for proteins. However the remaining 98.5% of non coding DNA can still play a regulatory role in protein synthesis which can affect the proteome. • Genes contain both regions of coding (extrons) and non coding (introns) DNA. The Pre-mRNA transcribed from genes is modified to remove introns and the extrons are joined back together in different ways. So one gene can form many different proteins. • Proteins can be modified after being synthesised. Synthetic Biology Science of making novel, artificial biological devices or organisms or the redesigning of current biological systems.

Sequencing DNA and the analysis of genomes allows for potential avenues for this. Examples: • Genetic engineering • Producing new organisms, proteins or cells • Storage and information via synthetic DNA (like Shakespeare) • Creation of biosensors e.g. for detecting pollutants • Exploiting naturally occurring proteins for biomaterials for nanotechnology • Creating immobilised enzymes. Genetic Engineering Genetically modified organisms (GMOs) are organisms which have had their genomes altered by genetic engineering techniques. They are developed to have desirable phenotypes. Transgenic Organisms Organisms that contain recombinant DNA- a DNA molecule that contains DNA from another organism. Works by taking a gene from one organism and inserting it into the genome of another. They can successfully express a gene from another organism as genetic code is universal. Process: 1) 2) 3) 4) 5)

Isolate Gene of interest from original organism Place gene inside a vector The vector insets the gene into a host cell Host cells that have successfully taken up the vector are identified The cells that express the gene of interest are grown or cloned

Isolating Desired Gene Using Restriction Endonuclease: - Enzymes that cut up DNA at recognition sites. - The specific sequences of bases are often palindromic meaning they read the same backwards as forwards - They are found in bacteria that use them to cut up viral DNA that have been inserted into the bacteria

- Each type of restriction endonuclease cuts up specific recognition sites in DNA - The gene of interest can be isolated by selecting the specific restriction endonuclease that cuts up recognition sites located near the gene - Some make clean straight cuts, others cut the two DNA strands unevenly, resulting in short regions of DNA with unpaired bases. Using Reverse Transcriptase: - Enzyme that converts RNA to DNA. - Found in retrovirus which stores genetic information in the form of RNA Process of using reverse transcriptase: 1. Cells that express the desired genes produce many strands of mRNA which are used as a template for synthesis of single-stranded complementary DNA (cDNA) 2. The mRNA template is hydrolysed with an enzyme to isolate a single strand of cDNA 3. Double stranded DNA is synthesised by DNA polymerase using the single stranded cDNA as a template 4. This contains the gene for the desired protein - The introns (non coding) have been already spliced out of the gene so can be correctly transcribed and translated by prokaryotic cells Using polynucleotide synthesiser: - The desired sequence of nucleotides can be determined by the primary structure of the desired protein which is fed into a computer which designs short sequences of nucleotides (oligonucleotides) that overlap with each other. - These get synthesised by the polynucleotide synthesiser in an automated process - The desired gene is assembled by joining together the overlapping oligonucleotides and is amplified using PCR - The polynucleotide is sent for DNA sequencing to make sure the process occurred without any errors. - The gene does not contain any introns (non coding DNA) so can be correctly transcribed and translated by prokaryotic cells ...