DNA Sequencing
___ is the enzyme that causes fireflies to glow. It uses luciferin and ATP as substrates, converting luciferin to oxyluciferin and releasing visible light.
Luciferase
____, which is still used for specialized purposes, such as analyzing DNA-protein interactions.
Maxam-Gilbert chemical degradation method
After the reaction has completed,___ is added to destroy any leftover dNTPs.
apyrase
Once a sequence is completed, it is usually analyzed by finding the genes and other features on it
annotation
Originally 2 methods of determining DNA sequence were invented around 1976, but only one is widely used: the ___ invented by Fred Sanger.
chain-termination method
Pyrosequencing: The light is detected with a ___ camera, similar to those used in astronomy.
charge-coupled device (CCD)
sequence a region, then make primers from the ends to extend the sequence. Repeat until the target gene was reached.
chromosome walking(aka primer walking) *how the CF gene was found
In DNA synthesis, a dNTP is attached to the 3' end of the growing DNA strand. The two phosphates on the end are released as ___.
pyrophosphate(PPi)
So, unlike ___, in Illumina Sequencing, you never have to worry about how many adjacent bases of the same type are present.
pyrosequencing
454 Technology: The ___ are attached to much smaller beads, which are then added to each well.
pyrosequencing enzymes
454 Technology: To start, the DNA is sheared into 300-800 bp fragments, and the ends are "polished" by ___.
removing any unpaired bases at the ends
454 Technology: The plate is then ____ with the each of the four dNTPs, plus other necessary reagents, in a repeating cycle.
repeatedly washed
Automated sequencers use 4 different fluorescent dyes as tags attached to the dideoxy nucleotides and run all 4 reactions in the ___.
same lane of the gel
metagenomics
sequencing DNA extracted from environmental samples
Illumina Sequencing Chemistry uses the basic Sanger idea of "____" of the second strand of a DNA molecule.
sequencing by synthesis
Sanger Sequencing: DNA polymerase always adds new bases to the ___ end of a primer that is base-paired to the template DNA.
3'
In Illumina Sequencing Chemistry, The fluorescent tags block the ___ of the new nucleotide, and so the next base can only be added when the tag is removed.
3'-OH
Pyrosequencing: The pyrosequencing machine cycles between the 4 dNTPs many times, building up the complete sequence. About ___ of sequence is possible (as compared to 800-1000 bp with Sanger sequencing).
300 bp
Illumina Sequencing the cycle is repeated ___ times
50-100
Sequencing reactions usually produce about ___ of good sequence.
500-1000 bp
___ uses PPi and adenosine 5'-phosphosulfate to make ATP.
ATP sulfurylase
Sanger Sequencing is done by having 4 separate reactions, one for each ___.
DNA base
In each reaction, __ starts creating the second strand beginning at the primer.
DNA polymerase
Sanger Sequencing uses ___ to synthesize a second DNA strand that is labeled.
DNA polymerase
Submission of the annotated sequence to___ allows everyone access to it: the final step in the scientific method.
Genbank
This idea is to put 2 different adapters on each end of the DNA, then bind it to a slide coated with the complementary sequences for each primer. This allows "bridge PCR", producing a small spot of amplified DNA on the slide.
Illumina Massively Parallel System
454 Technology:___ is added to the beads and an emulsion is created. PCR is then performed, with each aqueous droplet forming its own micro-reactor. Each bead ends up coated with about a___ identical copies of the original DNA.
Oil; million
_____ of random DNA fragments necessarily misses some regions altogether. Also, for sequencing methods that involve cloning (Sanger), certain regions are impossible to clone: they kill the host bacteria.
Shotgun sequencing
454 Technology: One adapter contains ____, which binds to a streptavidin-coated bead. The ratio of beads to DNA molecules is controlled so that most beads get only a single DNA attached to them.
biotin
The DNA bands fall into a ladder-like sequence, spaced one base apart. The actual sequence can be read from the ___.
bottom of the gel up
Today's sequencers use __ electrophoresis instead of__ gels.
capillary; slab
All 4 Sanger Sequencing reactions contain the 4 normal dNTPs, but each reaction also contains one of the ___.
ddNTPs
Sanger Sequencing also uses chain terminator nucleotides: ___, which lack the -OH group on the 3' carbon of the deoxyribose. When DNA polymerase inserts one of these___ into the growing DNA chain, the chain terminates, as nothing can be added to its 3' end.
dideoxynucleotides (ddNTPs)
The newly synthesized DNA from the 4 reactions is then run (in separate lanes) on an ___.
electrophoresis gel
454 Technology: The plate is coupled to a ___. A CCD camera records the light flashes from each well.
fiber optic chip
In shotgun sequencing, It is necessary to close gaps between contigs, and to re-sequence areas with low quality scores, this is a process called __?
finishing
Illumina Massively Parallel System slide contains millions of individual DNA spots. The spots are visualized during the sequencing run, using the__ of the nucleotide being added.
fluorescence
Illumina Sequencing Chemistry starts with a primer, new bases are added one at a time, with ____ used to determine which base was added.
fluorescent tags
Pyrosequencing: The ___ are added one at a time, with apyrase degradation and washing in between.
four dNTPs
For large genomes, ___ is a useful technique: first break up the genome into an ordered set of cloned fragments (scaffolds), usually BAC clones. Each BAC is shotgun sequenced separately.
hierarchical shotgun sequencing
Pyrosequencing: The amount of __ released is proportional to the number of ___ added. Thus, if the sequence has 2 A's in a row, both get added and twice as much light is released as would have happened with only 1 A.
light; bases
Radioactive nucleotides (32P) are used for __ sequencing.
non-automated
454 Technology: After the emulsion PCR has been performed, the oil is removed, and the beads are put into a "___" plate. Each well is just big enough to hold a single bead.
picotiter
Applications of Next Generation Sequencing
sequencing of whole bacterial genomes in a single run and genomes of individuals; metagenomics, looking for rare variants in a single amplified region, in tumors or viral infections; transcriptome sequencing
DNA is fragmented randomly and enough fragments are sequenced so each base is read 10 times or more on average. The overlapping fragments ("reads") are then assembled into a complete sequence.
shotgun sequencing
454 Technology: Adapters are added to each end. The DNA is made __ at this point.
single stranded
In the Sanger Sequencing reaction, the template DNA is usually ___
single stranded DNA
transcriptome sequencing
total cellular mRNA converted to cDNA.