Molecular Biology BIOL-4320-8

DNA sequencing

technique for determining the exact sequence of nucleotides, or bases, in a DNA molecule

What are different reasons why we would want to know the sequence of a DNA molecule

-To identify someone

-Identify bacteria

-Create a primer

-Identify a genetic disease

-Personalized medicine

-Genetic modification: Need to know sequence to insert or delete a gene

-Among others

How has DNA sequencing improved over time?

Human genome project took 10 years and cost almost $3 billion. Next-gen sequencing can now sequence a human genome in one day for just under $5,000

What happens during Sanger Sequencing?

DNA polymerase replicated DNA during sequencing reactions [sequence by synthesis], using a radiolabeled primer, dNTPs and ddNTPs

Dideoxynucleoside triphosphates [ddNTPs]

Nucleotide analogs, are used to interrupt DNA synthesis

What are the four different reactions performed for each ddNTP?

-ddATP

-ddCTP

-ddGTP

-ddTTP

What is different between a ddNTP and a normal dNTP

Normal dNTP has OH on 3’ and ddNTP has H on 3’

What does the 3’ OH on normal dNTPs do?

allows for the addition of nucleotides, without it, no nucleotides can be added

How is Sanger Sequencing done today?

-Each ddNTP labeled with different fluorescent dye

-Sequencing creates fragments of different lengths that are separated by capillary gel electrophoresis

-Laser detects fluorophore color to read DNA sequence

-Resulting DNA sequence plotted by computer as an electropherogram

-Generates read lengths of ~800-1,000 bp

What are the advantages of sanger sequencing?

-Allows to read through a whole set of genes/nucleotides

-Cheap to do

-Can accurately read sequences up to ~800-1,000 base pairs

What needs to be done to sequence a genome?

-Break genome into smaller DNA fragments

-Each fragment ligated into a vector

-Transform each vector into bacteria and select for transformants

-perform sequencing reactions using vector-specific primers

Contig

Series of overlapping clones or a continuous sequence defining an uninterrupted section of a chromosome

Sequence depth/sequencing coverage

Number of times that a particular nucleotide in the genome is sequenced on average

What is the general idea of shotgun sequencing

Take a genome with multiple copies, break it and the copies into random pieces, take pieces and search for overlaps [contigs], and then reassemble

What are the different types of Next Generation Sequencing?

-Emulsion PCR

-Solid Phase Bridge Amplification

What are different types of Emulsion PCR and what do they detect?

-454 pyrosequencing [detects light]

-Ion torrent [detects changes in pH]

What is a type of solid phase bridge amplification?

Illumina [reversible terminator sequencing]

What does each next-gen sequencing have in common?

-Need to extract and fragment DNA

-Synthetic oligonucleotides are ligated to all fragments

-Immobilization and PCR amplification of fragment [DNA clusters]

-Parallel sequencing of fragments

-Data captured by computer

What is a benefit of next-gen sequencing?

Efficient: millions of DNA clusters are sequenced at the same time and data capture is automated

What is the read length of 454 Pyrosequencing

400-500 nucleotides

How does 454 pyrosequencing work?

-Addition of complementary nucleotide by DNA polymerase releases pyrophosphate, which is converted into ATP by sulfurylase

-Luciferase catalyzes a reaction with luciferin and ATP that produces light flashes

-DNA sequence determined by detecting intensity of light flashes

What is a disadvantage of 454 pyrosequencing?

Cannot always distinguish how many bases are in a run of identical bases

What is the length read for Illumina sequencing?

100-200 bp

What happens in Illumina sequencing?

-DNA fragments attach to surface-bound oligonucleotides ha are complimentary to library adapters

-Attached fragments amplified through bridge amplification

-Leads to clusters of identical DNA strand, increasing signal strength