Gene Sequencing Flashcards
Describe and explain what gene sequencing is?
-The process of determining the nucleic acid sequence in DNA
-It includes any method of technology that it used to determine the order of the 4 bases
What was the Human Genome Project?
An international scientific research project with the goal of determining the sequence of nucleotide base pairs that make up human DNA, and of identifying and mapping all of the genes of the human genome
What was Fred Sanger’s DNA sequencing approach?
-to use a single strand of DNA as a template for 4 experiments in each dish
-a modified version of one of the DNA bases was added to the dish
-each modified base was labelled with a radioactive isotope, and once binded no more bases could be added
In 1980 how was Sanger’s sequencing method modified and why?
-Instead of using radioactive terminator bases, fluorescent ones were used instead, with each base being labelled with a different colour fluorescent marker
-it is much safer for scientists
-coloured nucleotides are also doubly de-oxidised
What is the basic principle of this PCR technique?
If these special bases attach to the fragments of DNA being copied in the PCR process, the DNA strand cannot grow any longer, even if it isn’t fully complete
Explain the process of PCR technique
1) multiple copies of the DNA to be sequenced are needed-done using normal PCR
2) the DNA is then broken into shorter pieces (2000-10000) bp long, or even smaller. This is known as shot gun fragmentation
3) these fragments are made single stranded, and then mixed with all the requirements for copying again (specific primers, DNA polymerase, free nucleotides some normal and some terminator), using modified PCR.
4) each fragment of DNA is copied up to the point where a terminator coloured nucleotide joins, then growth of the DNA molecule stops
5) this produces pieces of DNA of different lengths, depending upon the point where copying stops
6) these lengths of DNA can then be separated by Capillary tube Electrophoresis, with the shorter DNA strands travelling the furthest
7) a computer records the colours as they pass the end of the capillary tube. The first colour to pass represents the base at the end of the shortest fragment, the last colour to pass represents the base at the end of the longest fragment
Give an example of an extremely fast method of sequencing
Next generation sequencing
-very efficient and fast, so the cost of DNA sequencing has fallen greatly
Explain what pyrosequencing is and how it works
-uses activated nucleotides with phosphate attached to sequence the DNA
-start with a large piece of DNA, which is broken up into DNA fragments, which are then made single stranded.
-Sequencing primer is added, and then DNA is incubated with various enzymes e.g DNA polymerase and one of four activated nucleotides.
-The activated nucleotide joins to the DNA strand with help from DNA polymerase, and 2 phosphates and released and make ATP, and this activates another enzyme, leading to light being released.
-The amount of light generated is proportional to the amount of ATP available, which is proportional to how many types of activated nucleotide joins onto the newly formed DNA strand
Explain how gene sequencing has allowed scientists to compare individuals DNA, and why this is good.
-analysing the DNA sequence of an organism is a much more accurate way to decide which species an organism belongs to (classification)
-a useful method is to compare sections of a genome that are common to all species, but vary slightly. E.g cytochrome-c-oxidase
-this section of DNA is small enough to sequence quickly and cheaply, but varies enough to give clear differences between species
-the more similar the DNA base sequence for this enzyme, the more closely related the 2 species are
Explain how gene sequencing has allowed scientists to carry out proteomics and why this is good
-if researchers have an organisms genome printed out, they can use their knowledge of which base triplets code for which amino acid to determine the primary structure of a protein
-if scientists can work out the primary structure of a protein, they can then predict how it will arrange itself into the secondary and tertiary structures, allowing predictions on final specific 3D shape
-such developments have led to modified theories about how genes code for proteins; we now know there are 25000 coding genes in human DNA, but up to 1 million different proteins
-the study of proteomics is constantly increasing our knowledge of the complex relationship between genotype and phenotype
Explain how gene sequencing has allowed scientists to develop synthetic biology and why this js good
-it is the design and construction of new biological parts and systems, and the redesign of existing biological systems for purposes useful to humans
-one particular development is the ability of scientists to create DNA sequences in the lab from scratch. With the help of computers and laboratory chemicals organisms can be ‘designed’ to do new things e.g produce biofuels or drugs
What is bioinformatics and how does synthetic biology involve it?
-the incorporation of biology with computer science and statistics. Biological data is collected, organised, manipulated, analysed and stored. Large databases are created, ranging from genome sequence information to the amino acid sequences of proteins. Huge amounts of data can now be shared on a world wide level
