DNA Sequencing and Gene Analysis NIU Department 基因测序和基因分析牛科.pptVIP

* * * * * * * * * * * DNA Sequencing Determining DNA Sequence Originally 2 methods were invented around 1976, but only one is widely used: the chain-termination method invented by Fred Sanger. The other method is Maxam-Gilbert chemical degradation method, which is still used for specialized purposes, such as analyzing DNA-protein interactions. More recently, several cheaper and faster alternatives have been invented. It is hard to know which of these methods, or possibly another method, will ultimately become standard. We will discus two of them: 454 pyrosequencing and Illumina/Solexa sequencing Sanger Sequencing Uses DNA polymerase to synthesize a second DNA strand that is labeled. DNA polymerase always adds new bases to the 3’ end of a primer that is base-paired to the template DNA. DNA polymerase is modified to eliminate its editing function Also uses chain terminator nucleotides: dideoxy nucleotides (ddNTPs), which lack the -OH group on the 3 carbon of the deoxyribose. When DNA polymerase inserts one of these ddNTPs into the growing DNA chain, the chain terminates, as nothing can be added to its 3 end. Sequencing Reaction The template DNA is usually single stranded DNA, which can be produced from plasmid cloning vectors that contain the origin of replication from a single stranded bacteriophage such as M13 or fd. The primer is complementary to the region in the vector adjacent to the multiple cloning site. Sequencing is done by having 4 separate reactions, one for each DNA base. All 4 reactions contain the 4 normal dNTPs, but each reaction also contains one of the ddNTPs. In each reaction, DNA polymerase starts creating the second strand beginning at the primer. When DNA polymerase reaches a base for which some ddNTP is present, the chain will either: terminate if a ddNTP is added, or: continue if the corresponding dNTP is added. which one happens is random, based on ratio of dNTP to ddNTP in the tube. However, all the second strands in, say