Sanger sequencing Flashcards
Sanger sequencing
-also known as the chain termination method, is a technique used to determine the order of nucleotides (A, C, G, T) in a DNA molecule.
What does the fluorescence of ddNTP help with during DNA sequencing?
Fluorescent labeling of ddNTP helps in identifying specific bases during DNA sequencing by making them light up under a laser, aiding in determining the sequence of DNA strands.
Sanger Sequencing
-is a method for determining the nucleotide sequence of DNA.
-Named after Frederick Sanger, who developed the technique in 1977.
-It is also known as dideoxy sequencing or chain-termination sequencing.
Key Components of Sanger:
- DNA Template: The DNA fragment to be sequenced.
- Primers: Short single-stranded DNA molecules that initiate the sequencing reaction.
- DNA Polymerase: Enzyme that synthesizes new DNA strands.
- Nucleotides: Four standard deoxynucleotides (dNTPs: A, T, C, G).
- Dideoxynucleotides (ddNTPs): Modified nucleotides that terminate DNA synthesis (labeled with fluorescent dyes for detection).
Sanger Procedure:
- Preparation: DNA template is mixed with primers, DNA polymerase, dNTPs, and a small amount of ddNTPs.
2.Synthesis: DNA polymerase extends the primer by adding dNTPs, but incorporation of a ddNTP stops further extension.
- Fragment Generation: Produces DNA fragments of varying lengths, each ending with a ddNTP.
- Separation: DNA fragments are separated by size using capillary electrophoresis.
- Detection: Fluorescent labels on ddNTPs are detected, and the sequence is determined based on the color and length of the fragments.
Sanger Applications:
-Genetic Research: Identifies mutations, single nucleotide polymorphisms (SNPs), and small insertions/deletions.
-Medical Diagnostics: Detects genetic disorders and confirms mutations identified by other methods.
-Molecular Biology: Validates cloning and constructs in research.
Sanger Advantages:
-Accuracy: Highly accurate for determining DNA sequences up to about 1000 base pairs.
-Simplicity: Straightforward and reliable method.
-Gold Standard: Often used to validate sequences obtained by other methods.
Sanger Limitations:
-Length: Limited to relatively short DNA fragments (up to ~1000 base pairs).
-Throughput: Slower and less efficient for large-scale sequencing projects compared to next-generation sequencing (NGS).
-Cost: More expensive per base pair than NGS for large volumes of data.
Sanger Conclusion:
Sanger sequencing remains a fundamental and widely-used technique in molecular biology for its accuracy and reliability.
While it has been largely supplanted by next-generation sequencing for large-scale projects, it is still invaluable for specific applications such as validation of NGS results, sequencing of small regions, and diagnostic testing.
What is an X-linked disorder?
X-linked recessive inheritance refers to genetic conditions associated with mutations in genes on the X chromosome.
A male carrying such a mutation will be affected, because he carries only one X chromosome.
A female carrying a mutation in one gene, with a normal gene on the other X chromosome, is generally unaffected.
