Lecture 9: Next generation DNA sequencing Flashcards
What is DNA?
DeoxyriboNucleic Acid
What are the 4 nitrogous bases found in DNA?
adenine (A)
thymine (T)
guanine (G)
cytosine (C)
What are pyrimidines?
Pyrimidines include cytosine and thymine. A pyrimidine has a six-member nitrogen-containing ring, like purines, but no corresponding five-nitrogen ring.
What are purines?
Purines include adenine and guanine. A purine is composed of a six-member nitrogen-containing ring and a five-member nitrogen-containing ring joined together.
Describe the structure of DNA
Nucleotides are attached together to form two long strands that spiral to create a structure called a double helix. Nucleotides in the 2 opposing strands form hydrogen bonds to help secure the helix structure; adenine (A) always pairs with thymine (T) while cytosine (C) always pairs with guanine (G).
describe the structure of nucleotides
Combination of a nitrogen base, a sugar called deoxyribose and a phosphate group.
What is the charge of DNA (and RNA)?
Negatively charged
Describe the Maxam & Gilbert Method for DNA sequencing
Nucleobase-specific partial chemical modification of DNA and subsequent cleavage of the DNA backbone, at sites adjacent to the modified nucleotides, by hot piperidine. A series of labelled fragments is generated and the fragments are electrophoresed for size separation. From presence and absence of certain fragments the sequence may be inferred.
Describe the Snager Method for DNA seqencing
Selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. A test tube containing a single stranded DNA template is incubated with a primer, short oligonucleotide sequence specific to the DNA template, and DNA polymerase. The primer is used as a starting for the synthesis of DNA by polymerases in the 5’ to 3 ’direction. All deoxynucleotides (A, T, C and G) are added to the test tube, and a single type of dideoxynucleotide is added.
Why are dideoxynucelotides important for chain termination of DNA sequencing?
DNA synthesis requires a free 3’ hydroxyl on the deoxynucleotide; dideoxynucleotides lack this free 3’ hydroxyl group so prevent further nucleotides for being added and terminate DNA synthesis and polymerisation at various different points.
Why is the capillary fluorecesnt Sanger method preffered over manual Sanger method?
Capillary method is faster, cheaper and allows for longer sequences to be read.
What are the 3 examples of next-generation sequencing (NGS)?
- Roche 454 sequencing
- Illumina (Solexa)
- Ion torrent; Proton/ PGM sequencing
Describe the Roche 454 sequencing process
Uses the addition of nucleotides to fragments attached to beads in a flow cell. Detection of base addition by fluorescence; sequence can then be determined computationally from the signal density in each wash.
Masively parralel sequencing by synthesis.
Describe the Illumina sequening process
). Randomly fragmented DNA samples are attached to a ultra-high-density flow cell, where > 100 million DNA fragments are each amplified to a few thousand copies to form ‘clusters’. Adapters are added to the DNA fragments to enable ligation to the flow cell. After each round of sequencing, each cluster generates fluorescence of a colour specific to the base that has just been added. Computer then assembles the cluster of sequences, by integrating the series of fluorescent colours produced, into a longer read, often using a reference sequence as a template to enable identification of variants.
Masively parralel sequencing by synthesis.
Describe the Ion Torrent sequencing process
Each hole in the cell flow has a unique DNA molecule attached, from which the sequence is determined by adding a nucleotide by a phosphodiester bond. Detection of phosphodiester bond formation which results in the release of a proton and a change in pH. Computer detects change in pH and can identify nucleotide added to determine the DNA sequence.
Masively parralel sequencing by synthesis
