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.