![]() ![]() A current is passed through a gel, pulling the negatively charged DNA molecules through to the positively charged end of the gel. The newly made DNA fragments can be separated according to their size by gel electrophoresis. Separating and analysing the new DNA strands The end result is a whole lot of new DNA fragments, of varying length, all ending with a dideoxynucleotide. Because there are many more ordinary nucleotides than dideoxynucleotides, some chains will be several hundred nucleotides long before a dideoxynucleotide is added. The synthesis of new DNA is terminated when one of the dideoxynucleotides is added to the strand. The chemical change in a dideoxynucleotide, however, means that no additional nucleotides can be added, hence the name ‘terminator nucleotides’. With normal DNA nucleotides, one nucleotide can be attached to another and so on, forming a chain. The dideoxynucleotides are just like ordinary DNA nucleotides except that one hydroxyl (OH) group has been chemically changed to a hydrogen (H). Because all four ordinary DNA nucleotides are present in large amounts, the chain elongation continues normally – until by chance a dideoxynucleotide (terminator) is added in the place of a normal DNA nucleotide. The new DNA strand is made by complementary base pairing with the original DNA template. ![]() The primers anneal to the template strand, and the DNA polymerase enzyme makes a new strand of DNA by creating a complementary sequence of nucleotides drawn from the reaction mixture. One of the original DNA strands is used as a template for the synthesis of new DNA. buffer – to maintain the required pH for the reaction.DNA primers – short pieces of DNA that have been specially selected to bind to the selected DNA and are needed to get the reaction going.a mixture of terminator dideoxynucleotides (d dATP, d dGTP, d dTTP and ddCTP).a high concentration of normal DNA nucleotides (deoxynucleotides, d ATP, d GTP, d TTP and d CTP).Next, heat is used to separate the double-stranded DNA molecules into single strands. This is the process of creating multiple copies and is generally done using a process called PCR. The DNA to be sequenced must first be broken into smaller pieces and amplified. ![]()
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