Module 5 Flashcards
Sanger sequencing
- Primer with 3’ end base pairs to a single stranded DNA template
- Generate nested DNA fragments that vary by only 1 nucleotide
- Nested fragments are sorted by size
- Fragments start with a common 5’ sequence
ddNTP
A dNTP that is missing the 3’ OH group and cannot form phosphodiester bonds
PAGE
PolyAcrylamide Gel Electrophoresis allows for high resolution separate by fragment size
Automated sanger sequencing
Fluorescent dyes are attached to ddNTPs to replace radioactive labels
Parallelism
Many different reactions run at once
Throughput
Amount of sequence information generated in a specific time
Next generation sequencing technologies steps
- template preparation
- sequencing chemistry and detection
- data analysis
Illumina
Sequencing by synthesis, massively parallel, uses reversible fluorescently labeled ddNTPs
Clonal bridge amplification
An oligo complimentary binds to an adapter region and forms a bridge where polymerase turns it into a double stranded bridge, creating two oligo strands
Paired ends
An application that sequences the ends of a DNA fragment
SMRT
Single molecule real time sequencing harnesses the natural power of DNA polymerase activity and detects DNA synthesis in real time
Nanopore
A protein with a hole in the middle that is set in an electrically resistant membrane
Bioinformatics
The intersection of biology, computer science, and informational tools
Chromatogram/Electropherogram
Output sequence reads
Phred
Base-calling algorithm used to interpret and check quality of sequence reads. Calculates the likelihood that a base call is incorrect
