DNA sequencing

Question 1

what is DNA sequencing

Answer 1

the process of determining the precise order of nucleotides within a DNA molecule

Question 2

how did Sanger develop DNA sequencing

Answer 2

• using viruses and bacteria
• radioactive labelling of bases and gel electrophoresis on a single gel
• it was initially manual so took a long time but eventually in 1970s, Sanger could read 500-800 bases at a time using Sanger sequencing

Question 3

how has Sanger sequencing developed

Answer 3

• swapping of radioactive labels for coloured fluorescent tags which led to scaling up and automation of the process
• led to the capillary sequencing version of the Sanger sequencing method that was used during the Human Genome Project and similar techniques are used today

Question 3

when was the Human Genome Project established

Answer 3

• 1990
• involved sequencing the DNA of smaller, simpler organisms to refine and develop the techniques
• aim was to complete the HGP project in 15 years but the automation of sequencing techniques and development of more powerful, faster computers, meant that the first draft of the human genome was ready in 2000
• first complete human genome sequence was published in 2003, 2 years ahead of schedule

Question 4

terminator bases

Answer 4

• modified versions of the 4 nucleotide bases ATCG which stop DNA synthesis when they are included
• an A terminator stops synthesis at the location that an A base would be added and so on
• the terminator bases are given coloured fluorescent tags

Question 5

capillary method of DNA sequencing process

Answer 5

• DNA, primer, DNA polymerase, excess of normal nucleotides, terminator bases are mixed
• in thermal cycler which rapidly changes temperature at programmed intervals repeatedly
• DNA double strand separates, primers anneal and polymerase builds up new DNA strands

Question 6

terminator bases’ role in DNA sequencing process (capillary method)

Answer 6

• each time a terminator base is incorporated instead of normal nucleotide, synthesis of DNA is terminated so no more bases are added
• chain-terminating bases are present in lower amounts and added at random
• results in many DNA fragments of different lengths depending on where the chain terminating bases have been added during the process
• after many cycles, all possible DNA chains will be produced with the reaction stopped at every base
• DNA fragments are separated according to their length by capillary sequencing
• order of bases in capillary tubes shows the sequence of new complementary strand of DNA so used to build up sequence of og strand

Question 7

capillary sequencing

Answer 7

• works like gel electrophoresis in minute capillary tubes
• fluorescent markers on the terminator bases are used to identify the final base on each fragment
• lasers detect the different colours and thus the order of the sequence

Question 8

what happens with data from the sequencing process

Answer 8

• data from sequencing process is fed into computer that reassembles genomes by comparing all the fragments and finding areas of overlap between them
• once a genome is assembled, scientists identify the genes that code for specific characteristics
• medical researchers want to identify regions that are linked with particular diseases

Question 9

next generation sequencing

Answer 9

• using Sanger sequencing, working out base sequence of short strands of DNA was difficult and time-consuming
• DNA tech have become faster and more automated, leading to high-throughput sequencing process
• reaction takes place on plastic slide (flow cell) instead of gel/capillaries
• millions of fragments of DNA are attached to the slide and replicated in situ using PCR to form clusters of identical DNA fragments
• still uses coloured terminator bases to stop the reaction so images can be taken
• all clusters are being sequenced/imaged at the same time so known as massively parallel sequencing
• lower cost, so more genomes can be sequenced

Question 10

bioinformatics

Answer 10

the development of software and computing tools needed to organise and analyse raw biological data, including the development of algorithms, mathematical models, and statistical tests that help us make sense of the enormous quantities of data being generated

Question 11

computational biology

Answer 11

• study of biology using computational techniques, especially in the analysis of huge amounts of biodata
• uses data from bioinformatics to build theoretical models of biological systems which can be used to predict what will happen in different circumstances
• e.g. important in the analysis of the data from sequencing billions of base pairs in DNA, working out 3D protein structures, understanding molecular pathways like gene regulation
• helps use information from DNA sequencing e.g. identifying genes linked to specific diseases in populations and in determining the evolutionary relationships between organisms

Question 12

analysing human genome using computational biology

Answer 12

• computers can analyse and compare the genomes of many individuals, revealing patterns in the DNA we inherit and the diseases to which we are vulnerable
• enormous implications for health management and medicine
• however with the exception of a few relatively rare genetic diseases caused by changes in a single gene, our genes work together with the environment to affect our physical characteristics, physiology and susceptibility to disease

Question 13

real world implications for analysing genomes of pathogens

Answer 13

become fast and relatively cheap which leads to:
- doctors find source of infection e.g. bird flu/MRSA
- doctors identify antibiotic-resistant strains of bacteria ensuring antibiotics will only be used when they will be effective to prevent spread of antibiotic resistance e.g. TB, HIV/AIDS
- scientists track progress of an outbreak of a potentially serious disease and monitor potential epidemics e.g. flu each winter, Ebola
- scientists identify regions in the genome of pathogens that may be useful targets in the development of new drugs and to identify genetic markers for use in vaccines

Question 14

identifying species using DNA barcoding

Answer 14

• difficult to determine the species of an organism using traditional observation
• genome analysis aids species identification by comparing to standard sequence for the species
• one technique involves identifying particular sections of the genome common to all species but vary between them, so comparisons can be made (DNA barcoding)
• in the iBOL project, scientists identify species using relatively short sections of DNA in the gene cytochrome c oxidase (coding for enzyme involved in cellular respiration)
• small enough section to be sequenced quickly and cheaply, yet varies enough to give clear differences between species
• in land plants, that DNA region doesnt evolve quickly enough to show clear differences between species, but 2 regions in chloroplast DNA have been identified which can be used for species identification
• no suitable regions for fungi and bacteria yet

Question 15

DNA sequencing in evolutionary relationships

Answer 15

• DNA sequences for can be compared between organisms
• because basic mutation rate of DNA can be calculated, scientists can calculate how long ago 2 species diverged from a common ancestor
• enables evolutionary trees to be built with high accuracy

Question 16

proteomics definition

Answer 16

the study and amino acid sequencing of an organism’s entire protein complement

Question 17

genomics and proteomics

Answer 17

• scientists thought that each gene codes for one protein
• but now know there are 20-25000 coding genes in the human DNA and a different number of unique proteins
• DNA sequence should predict sequence of amino acids in the proteins it produces
• but evidence that sequence of amino acids is not always as predicted by the genome sequence alone as some genes code for many different proteins
• spliceosomes and protein modification

Question 18

spliceosomes

Answer 18

• mRNA transcribed from DNA in nucleus includes both exons and introns
• before it lines up on the ribosomes to be translated, the pre-mRNA is modified
• introns are removed, and maybe some exons
• then exons to be translated are joined together by enzyme complexes called spliceosomes to give mature functional mRNA
• spliceosomes may join the same exons in variety of ways so single gene may produce several versions of functional mRNA which could code for different arrangements of amino acids, giving different proteins and phenotypes

Question 19

protein modification

Answer 19

• some proteins are modified by other proteins after they are synthesised
• a protein coded for by a gene may remain in tact or shortened or lengthened to give another protein
• study of proteomics is constantly giving increasing knowledge of the complex relationship between genotype and phenotype

Question 20

synthetic biology

Answer 20

• the design and construction of novel artificial biological pathways, organisms, or devices, or the redesign of existing natural biological systems
• the ability to sequence genome of organisms and understand how each sequence is translated into amino acids
• along with increasing ability of computers to store, manipulate and analyse the data

Question 21

what techniques does synthetic biology include

Answer 21

• genetic engineering (single change in biological pathway or relatively major genetic modification of entire organism
• use of biological systems/ parts in industrial contexts (immobilised enzymes and production of drugs from microorganisms)
• synthesis of new genes to replace faulty genes (developing CF treatments: synthesising functional genes in labs and using them to replace faulty genes in cells)
• synthesis of entirely new organism (scientists created artificial genome for bacterium and successfully replaced og genome with this new one