Dna Sequencing 3 Flashcards
What are the methods used to sequence DNA
Maxam- Gilbert
Sanger
Illumina-solexa
Oxford nanopores
How does maxam-Gilbert sequencing work roughly
Roughly how does Sanger sequencing work
What types of sequences do the DNA sequencing methods read
What is the outline for the Sanger sequencing process
DNA Fragment Preparation: The DNA fragment to be sequenced is first isolated and purified. This DNA can be obtained from various sources, such as PCR-amplified DNA, plasmids, or genomic DNA.
Primer Annealing: A primer complementary to a specific region of the DNA fragment is annealed to one end of the DNA fragment. This primer serves as the starting point for DNA synthesis.
DNA Polymerase Extension: DNA polymerase, along with nucleotides (dATP, dCTP, dGTP, and dTTP), is added to the reaction mixture. The DNA polymerase extends the primer by incorporating complementary nucleotides onto the template DNA strand. However, in Sanger sequencing, each reaction tube contains a small amount of a chain-terminating dideoxynucleotide (ddNTP), in addition to the regular nucleotides.
Incorporation of Chain-Terminating ddNTPs: The presence of the chain-terminating ddNTPs (ddATP, ddCTP, ddGTP, or ddTTP) causes DNA synthesis to terminate when they are incorporated into the growing DNA strand by DNA polymerase. These chain-terminating ddNTPs lack the 3’-OH group required for the formation of the phosphodiester bond, which prevents the addition of the next nucleotide.
Fragment Separation by Gel Electrophoresis: After the DNA synthesis reaction, the reaction mixture is subjected to gel electrophoresis. This separates the DNA fragments based on their size. Typically, the gel is made of polyacrylamide or agarose.
Visualization of Fragments: Once the DNA fragments are separated, they are visualized using a technique such as autoradiography or fluorescent imaging. In autoradiography, the gel is exposed to X-ray film, and radioactive labels on the DNA fragments generate an image on the film. In fluorescent imaging, fluorescently labeled nucleotides are used during DNA synthesis, and the DNA fragments are visualized using a fluorescent scanner.
Sequence Analysis: The DNA sequence is determined by analyzing the pattern of bands or peaks on the gel. Each band or peak corresponds to a different nucleotide in the sequence. The sequence is read from the bottom (5’ end) to the top (3’ end) of the gel.
Data Interpretation: The sequence data are analyzed and interpreted using bioinformatics tools and software. The individual nucleotide bases are identified based on their position in the gel and the color or intensity of the bands or peaks.
Assembly and Final Sequence: If necessary, the individual sequence reads are assembled into a contiguous sequence using sequencing software. The final DNA sequence is then obtained and can be further analyzed or used for various research or diagnostic purposes.
How is the DNA analysed after Sanger sequencing
What is cycle sequencing
In cycle sequencing, the DNA sequencing reaction is performed in a thermal cycling device, such as a PCR machine. The key components of the cycle sequencing reaction include:
Template DNA: The DNA fragment to be sequenced serves as the template for the sequencing reaction. This DNA can be single-stranded or double-stranded, depending on the specific sequencing protocol.
Primer: A short single-stranded DNA primer is annealed to the template DNA at a specific location. The primer serves as the starting point for DNA synthesis by a DNA polymerase enzyme.
DNA polymerase: A DNA polymerase enzyme, such as Taq DNA polymerase or a modified version of it, is used to catalyze the synthesis of new DNA strands complementary to the template strand.
Nucleotides: A mixture of deoxynucleotide triphosphates (dNTPs) is provided in the reaction, including dATP, dTTP, dCTP, and dGTP, which are the building blocks for DNA synthesis.
Dideoxynucleotides (ddNTPs): Each of the four dNTPs is labeled with a different fluorescent dye and modified to include a dideoxy group, which lacks a 3’-OH group. These ddNTPs act as chain terminators when they are incorporated into the growing DNA strand by the DNA polymerase.
What is a virtual electropherogram
Can be used after Sanger sequencing
How does illumina sequencing work
In short:
1 prepare DNA sample
2 attach DNA to surface
3 bridge amplification
4 fragments become double stranded
5 denature the double stranded molecules
6 complete amplification
7 determine the first base
8 image the first base
How does Pacific biosciences waveguide technology work to sequence DNA
How can oxford nanapores be used to sequence DNA
What is metagenamics
What are the challenges with genome sequencing
1 when do you know if a genome is fully sequenced
2 is one genome enough- is that genome representative of all the organisms
3 what is the function of the DNA sequences in the genome - this hasn’t been determined fully yet
