6.3 Manipulating Genomes Flashcards
What is DNA sequencing?
A technique that allows genes to be isolated and read, to determine the sequence of bases.
Outline Sangers approach
- use single strand of DNA as template for four experiments in separate dishes
- each dish contained a solution with four bases A, C, T, G, plus DNA polymerase
- to each dish, a modified version of one of the DNA bases was added, which were labeled with a radioactive isotope
- once this modified base was added into the synthesized complementary strand of DNA, no more bases could be added
- thousands of DNA fragments of varying lengths were generated
- passed through a gel by electrophoresis -> sorted into lengths
- nucleotide base at the end of each fragment was read according to its radioactive label
Outline high throughput/ pyrosequencing
- DNA cut into fragments using a nebuliser
- lengths degraded into single stranded DNA and these are immobilised
- sequencing primer is added
- only one of the four activated nucleotides (ATP, TTP, CTP, GTP) is added at any one time and any light generated is detected
- one activated nucleotide is incorporated into a complementary strand of DNA (help of DNA polymerase)
- as this happens, the two extra phosphate groups are released
- in presence of APS, ATP sulfurylase converts the pyrophosphate to ATP
- luciferase converts luciferin to oxyluciferin which generates visible light that can be detected by a camera
- the light generated indicates how many of the same type of activated nucleotide were incorporated adjacently into the complementary DNA strand
- apyrase degrades the unused nucleotides and the reaction starts again
What is bioinformatics?
Storage, retrieval and analysis of universal data from studies. Allows scientists to make comparisons with the genomes of other organisms using the databases available
What are the applications for gene sequencing?
1) comparisons between species, e.g. found only a few genes unique to humans. Many of differences between organisms are not because the genes are different, but because some of the shared genes have been altered and work in subtly different ways, or changes to regulatory regions
comparing genomes of organisms thought to be closely related helps to confirm their evolutionary relationships. DNA from bones and teeth of some extinct animals can be amplified and sequenced so that their history can be verified
2) allows for comparisons between individuals: although humans all have the same genes (mostly), our alleles differ due to mutations. Places on DNA where substitutions occur are called single nucleotide polymorphisms (SNPs). Methylation of certain chemical groups in DNA plays a major role in gene expression. Methods to map this in the epigenetics field can help us understand the development of certain diseases.
3) predicting amino acid sequences of proteins.
4) synthetic biology (designing and building useful biological devices and systems) -> e.g. production of drugs by genetically engineering bacteria or yeast, novel proteins, biosensors, and materials produced for nanotechnology.
What is the DNA profiling procedure?
- DNA obtained
- DNA digested with restriction enzymes, that cut the DNA at specific recognition sites. These will cut the DNA into fragments which vary in length between individuals
- Fragments separated by gel electrophoresis and stained. Larger fragments travel the shortest distance
- Banding pattern can be seen
- DNA to which the individual is being compared is treated with the SAME restriction enzyme and also done in electrophoresis.
- The banding patterns of the DNA can then be compared
What part of the DNA is analyzed?
Short tandem repeat sequences (in introns). These contain mini satellites and micro satellites
Highly variable short repeating lengths of DNA that are polymorphic, and the NUMBER varies from person to person (not the code)
What are the applications of DNA profiling?
- Forensic science: identify war criminals, establish innocent suspects, identify victims body parts after disasters, identify remains
- Maternity and paternity disputes: comparing DNA profiles of mother, father and child can establish maternity or paternity
- Analysis of disease: protein electrophoresis can detect type of haemoglobin present and aid diagnosis of sickle cell, as well as detect a varying number of repeat sequences for conditions such as Huntingtons.
What is the purpose of PCR?
biomedical technology that can amplify short lengths of DNA to thousands of millions of copies
Explain the denaturation step. Why?
Mixture is heated -> denatures DNA to break hydrogen bonds between two strands, to separate them, to expose the nucleotide bases.
Explain the annealing steps. Why are primers needed?
- Mixture cooled to around 68 degrees celsius so the primers can anneal to one end of each single strand of DNA
- Taq polymerase can now bind to end where there is double stranded DNA
Explain the extension steps. Why is the temp raised?
- Temperature raised to 72 degrees to keep the DNA as single strands and this is the optimum temperature for Taq polymerase
- Taq catalyses addition of DNA nucleotides to single-stranded DNA molecules, starting at the end with primer and proceeding in 5’ to 3’ direction.
- Taq reaches the other end of the DNA molecule, then a new double strand of DNA has been generated.
How do you calculate the number of DNA molecules after n cycles of PCR?
2^n
Compare and contrast DNA replication with PCR
Explain why PCR is used in DNA fingerprinting
