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DNA sequencing by Mind Map: DNA sequencing

1. Methods

1.1. Dideoxy seq

1.1.1. Principle DNA polymerase synthesizes complementary copies of single stranded DNA template When a 2’, 3’-dideoxynucleoside triphosphate (ddNTP) added into a growing strand -> Termination Random chain termination creates diff size fragments . DNA sequence is deduced from the pattern of DNA fragments generated by the incorporation of respective ddNTPs

1.1.2. Types Manual 4 rxns: EACH with 1 type of ddNTPs. DNA synthesis products are analyzed manually. DNA sequence is read manually low throughpt: ~300 bp per run Automated 1 rxn: 4 types of ddNTPs. DNA synthesis products are analyzed automatically. DNA sequence is generated automatically high throughput: up to 1,200 bp per rxn and 96 rxns every 3 hours with capillary sequencers.

1.2. Chemical degradation

2. Application

2.1. Confirmatory seq

2.1.1. For what? identify and map mutations by site-directed mutageneis verify structr and/or orientation of a recombint DNA construct

2.1.2. how? often required 1 set of rxn only approp restriction fragmt is subcloned into approp vector becuz "region of interest" usually lie within the sequencing range of a "universal primer", that anneals to a vector sequence just beyond the target DNA. if not, synthesize a primer of17-19 nucleotides long which anneals 50-100 bp from the region of interest.

2.2. De novo seq

2.2.1. for what? provide accurate nucleotide sequence of a segment DNA (may be several kb in length) with UNKNOWN seq

2.2.2. how? Primer walking Clone the gene into a vector Perform dideoxynucleotide sequencing reaction using one primer annealing to the vector near the DNA insertion site Identify the sequence of the first ~500 bases of the insert DNA And so on Generate nested deletions with exonuclease digestion Incubate template DNA with exonuclease or DNase I Remove an aliquot of digested product at a regular time interval Clone digested products separately into a vector by blunt-ended ligation Use universal primer to sequence the insert DNA Assembly sequences of all fragments in silico to deduce the sequence Shotgun cloning and sequencing Randomly fragment target DNA by sonication or digestion with DNase I Create blunt-end fragments by Phosphorylate 5’ ends with T4 DNA KINASE Short DNA fragments by size & clone into vectors Sequence the DNA fragments: each nucleotide site should be from different fragments sequenced 6-10 different times Pyrosequencing Based on the detection of pyrophosphate that is released when a phosphodiester bond forms during DNA synthesis A nu is incorporated to growing strand, releasing a pyrophosphate (PP) PP + Adenosine-5-phosphosulfate -ATP sulfurase ---> ATP ATP + Luciferin + Luciferase -> light + AMP + oxylucifrin Cyclic array sequencing Emulsified PCR DNA is attached to 2 adapters A and B, only B can bind to Biotin Filter with Streptavidin-coated beads: only adapter B can bind to. Now only A-B and B-B left take the A-B out to use by melting the beads, B-B stick to the beads A-B (5'-3') is amplified in oil drop (microreactor)

2.2.3. Maximum of 600-800bp of good DNA sequence possible in a single run