Biology (Year 12) - Biotechnology: Processes
DNA sequencing refers to the methods and technologies which are used in order to determine the order of nucleotide bases in a DNA molecule, and allows us to perform a thorough analysis of DNA as it provides us with highly comprehensive information: the nucleotide sequence. There may be a number of survival benefits in identifying the nucleotide sequence of DNA, for example, genes that increase drought resistance or salt tolerance in plants may be identified and studied. Sequencing genes of different species has also assisted scientists in determining genetic relatedness and evolutionary links. DNA sequencing used to be done manually, called Sanger sequencing, but is now done automatically in a process called next-generation sequencing. The Sanger method, also referred to as dideoxynucleotide sequencing or chain-termination sequencing, is based on the use of dideoxynucleotide triphosphates (ddNTPs) in addition to the normal triphosphates (dNTPs) found in DNA. ddNTPs are highly similar nucleotides, except they contain a hydrogen group (H) on the 3’ carbon instead of a hydroxyl group (OH). These modified nucleotides, when integrated into a DNA sequence, prevent the addition of further nucleotides, thus stopping the elongation of the DNA chain. This occurs as a phosphodiester bond cannot form between the dideoxynucleotide and the next incoming nucleotide and thus the DNA chain is terminated. 1. The region of DNA to be sequenced is identified, cut and amplified, then heated and denatured to produce single-stranded template DNA 2. Template DNA, primer, DNAP, all 4 types of dNTPs and one type of dyed ddNTP are added to the reaction mixture 3. The sequencing DNA primer is annealed to single-stranded DNA at 3’ end of the original strand, providing starting sequences for synthesis 4. DNA polymerase extends the new strand by attaching complementary dNTPs in the 5’ to 3’ direction 5. When a dideoxynucleotide that has been coloured with fluorescent dye attaches at random, the newly synthesised strand terminates (the ddNTP prevents the formation of phosphodiester bonds) 6. By performing four separate reactions, four separate sets of chain-terminated fragments are produced 7. Following termination, heating to denature the partially double-stranded molecules releases the single-stranded chain termination molecules of the various lengths from their templates 8. They can then be separated using gel electrophoresis – the nucleotides (different colours) run in separate lanes 9. As gel electrophoresis proceeds, a laser scans across the bottom of the gel, detecting the different dyes and revealing the base sequence – terminated strands line up from smallest to largest 10. The sequence of the original region of DNA is finally deduced by examining the relative positions of the dideoxynucleotide chain termination products in the four lanes of the denaturing gel Next-generation sequencing applies the same principles as the Sanger method, but the technology is more advanced and efficient. The three basic steps are DNA preparation, sequencing and analysing. 1. DNA preparation: DNA is isolated and purified, then cut into 300bp fragments. The fragments are then amplified using a PCR type method to create massive numbers of identical copies. The resulting fragments are single-stranded. The different types of fragments are placed into unique wells and barcoded. 2. Sequencing: The multi-well plate contains assorted fragments. In each well, modified versions of the four nucleotides wash over the mixture. The nucleotides hydrogen bond to the DNA template strand according to base-pairing rules. Each nucleotide has one of four fluorescent tags attached. The tags indicate the positions and thus the order of the four nucleotides. A terminating set of nucleotides is also in the mix, preventing further elongation. Each time a chemically tagged nucleotide attaches to the template strand, there is a flash of light and this is recorded. 3. Data analysis: The recorded light flashes reveal the sequence of nucleotides of the template strand in each well. The sequencing software identifies the nucleotides by the order of the colours recorded. In both NGS and Sanger sequencing, DNA polymerase adds fluorescent nucleotides one by one onto a growing template strand. Each incorporated nucleotide is then identified by its fluorescent tag, and one colour represents one nucleotide. The main difference between the sequencing methods is the number of DNA sequenced at once; one molecule versus many. The Sanger method only sequences one DNA fragment at a time, whereas NGS sequences hundreds to thousands of genes at one time.
A genome map (the act of gene mapping) involves identifying and recording the positions (different loci) of different genes, as well as the distances between the genes found on a chromosome; genome mapping means that all chromosomes in a species' somatic cell are mapped. A genetic marker plays a key role in gene mapping. A genetic marker is a gene or a sequence on a chromosome with a known location that is associated with a specific trait. Genetic markets may include a number of types of sequences of DNA (for example, short tandem repeats (STRs), variable nucleotide tandem repeats (VNTRs), regulatory genes etc.). A genetic marker is used as a distinguishable area on a chromosome which assists a biotechnologist to locate genes that cause specific traits such as disease or a desirable phenotype. Genetic markers are also useful in genome mapping, as when a gene exists on a chromosome in close proximity to a marker, there is a higher chance that the gene is more likely to stay together during crossing over (the distance between these two is known as 'linkage distance'), enabling a researcher to determine the relative location of the desired gene. DNA sequencing enables genome mapping to occur as the sequenced DNA fragments can be aligned to the genome map in order to aid with the assembly of the genome.