Ch. 10: DNA sequencing

name different types of DNA sequencing methods

Maxam/Gilbert chemical sequencing

Sanger chain termination sequencing

pyrosequencing

array sequencing

sequencing by ligation (next generation)

maxam/gilbert chemical sequencing

performed by chain breakage at specific nucleotides

resulting DNA fragments are separated by gel electrophoresis & read from bottom to top (5’ tp 3’)

at what direction are sequencing gels (results) read in Maxam/Gilbert chemical sequencing method?

bottom to top (5’ to 3’)

chain termination (sanger) sequencing

is a modified DNA replication

DNA sequencing method that relies on the selective incorporation of dideoxynucleotides (ddNTPs) by DNA polymerase. These ddNTPs lack a 3'-hydroxyl group, which is essential for further DNA extension, causing the DNA synthesis to terminate when a ddNTP is incorporated. This process generates a series of DNA fragments of varying lengths, each terminated by a ddNTP, which can then be separated and analyzed to determine the DNA sequence. `

what are ddNTPS & what are they used for?

** extra info - Deoxyribonucleoside Triphosphates (dNTPs) are natural building blocks for DNA synthesis

Dideoxyribonucleoside Triphosphates (ddNTPs) are MODIFIED versions of dNTPs designed to terminate/stop DNA chain elongation

they are used in chain termination (sanger) sequencing technique to stop DNA elongation

how does dideoxyribonucleoside triphosphates (ddNTPs) stop DNA elongation ?

ddNTPs LACK a 3”-OH group which prevents formation of phosphodiester bond which prevents the addition of next nucleotide & stops DNA chain from elongating

Sanger sequncing AKA chain termination sequencing - how it works?

1. sequencing reaction mix : primer + template

2. 4 ddNTPS labeled w/ different fluorescent dyes corresponding to a specific dNTPs (G/C/T/A; normal nucleotides)

3. ddNTPs modified (lack 3’-OH group) —> stop DNA synthesis creating DNA fragments of different lengths

4. DNA fragments (aka sequencing ladder) are separated by electrophoresis

5. fragments from each tube (1 tube for each ddNTP & corresponding dNTP) are placed in separate gel lanes —> fragments separated based on size

6. sequence read bottom to top (5’ to 3’)

what is cycle sequencing

method of DNA sequencing used in Sanger sequencing method that is performed in a thermal cycler

due to cycle sequencing being performed in a thermal cycler, what is requireed for the dna polymerase ?

dna polymerase must be HEAT STABLE

in dye primer sequencing, the primer containing fluorescent dye “labels” what part of the sequencing ladder ?

5’ ends

in dye terminator sequencing, the fluorescent dye is covalently attached to ? and “labels” what part of the sequencing ladder?

fluorescent dye attach to dideoxynucleotides

& “labels” 3’ end of sequencing ladder

in the dye terminator sequencing method, only 1 tube is needed for all 4 ddNTPs. What is used, for only 1 tube to be needed for all ddNTPS rather than 4 tubes for each ddNTP (commonly seen in sanger sequencing)?

specific dye is used for each ddNTP & is able to get distinguished by color & size —> allow for a single sequencing reaction

what is the difference b/w dye terminator sequencing vs sanger sequencing?

dye terminator sequencing a NEWER, MODERN AUTOMATED version of sanger sequencing

dye terminator requires only ONE gel lane/capillary vs. sanger sequencing need 4 gel lanes

how is the DNA ladder read in the dye terminator sequencing method?

electropherogram (electrophoresis based technique)

what do the peak represent on an electropherogram?

mutations or sequence changes

what is bisulfite sequencing ?

technique that studies DNA methylation (methyl added to cytosine base) —> uracil

how is uracil made in bisulfite sequencing ?

DNA treated w/ sodium bisulfite which convert unmethylated cytosines to uracil

is methylated cytosine treated with sodium bisulfite?

yes it is , but it is not “changed/affected” (stays as cytosine & is not converted to uracil)

when comparing treated cytosine in bisulfite sequencing, what are we looking for (goal/ what does it mean)?

compare treated & untreated DNA template then we look for uracil conversion in template = help determine which cytosines were methylated

Example:

if C (cytosine) —> U (uracil) = sequence/C was unmethylated

if C —> C = sequence/C was methylated b/c methylated C are not affected/changed w/ sodium bisulfite treatment

what is pyrosequencing ?

technique based on the generation of light signal through released pyrophosphate (ppi) on nucleotide addition.

DNA_n + dNTP —> DNA_n+1 + PP_I

in pyrosequencing, what is PPi and what is its function?

PPi (pyrophosphate) is released as a byproduct when nucleotide is incorporated into a growing DNA strand

function = generate ATP from adenosine phosphosulfate (APS)

APS + PPi —> ATP

how is light produced in pyrosequencing ?

PPi trigger enzymatic rxn: APS + PPi —> ATP —> luciferase use ATP (convert luciferin to oxyluciferin) —> produce light

in pyrosequencing, how many nucleotides will generate a light signal ?

¼ generate light signal

in pyrosequencing, how are the light signals recorded/measured?

pyrogram

what is array sequencing ?

high density oligonucleotide array with labeled sample DNA which hybridizes to synthetic array probes

in array sequencing, where is the complementary probe located ?

complementary probe located on the array

how are the results dipicted in array sequencing method?

graph

google definition of array sequencing

involves using a DNA chip (microarray) where thousands of tiny spots, each containing a specific DNA sequence (probe), are arranged in a grid. A sample of DNA is then added to the chip, and the probes bind to any complementary DNA sequences in the sample. The binding is detected by fluorescence, allowing researchers to identify which DNA sequences are present in the sample. 

google definition of next generation sequencing

revolutionary DNA sequencing technology that allows researchers to analyze vast amounts of genetic information quickly and cost-effectively. It differs from traditional Sanger sequencing by its ability to sequence millions of DNA fragments simultaneously, making it ideal for exploring whole genomes, exomes, and specific gene regions. 

what are some common examples of next generation sequencing technologies?

454 FLX Roche ; pyrosequencing

Illumina Cluster sequencing

SOLiD

what is the run time for next generation sequencing

10hrs - 5 days w

what is the length of bases that next generation sequencing can read?

35-400 bases

what is an example of next generation sequencing being used in the lab?

the human genome project