22.5-22.6

In Pyrosequencing, how are the nucleotides flowing across the plate in sequencing?

1 at a time, when the light flashes means that the nucleotide has been incorporated

What is a metagenome?

A collection of genes from an environmental sample

Next-generation sequencing technologies

Ability to process thousands or even millions of sequence reads in parallel

PacBio SMRT: DNA Prep Enzymes Used Detection

DNA Prep - DNA fragments and DNA polymerase are trapped within tiny holes on a thin metal film Enzymes - DNA Polymerase Detection - Growth of individual DNA molecules is monitored by fluorescence imaging

Illumina: DNA Prep Enzymes Used Detection

DNA Prep - DNA fragments are bound to a flow cell surface Enzymes used - DNA Polymerase Detection - Four different fluorescently labeled nucleotides are detected

Pyrosequencing: DNA Preparation Enzymes Used Detection

DNA Prep - DNA fragments bound to small beads, which are dropped into tiny wells in a picotiter plate Enzymes used - DNA polymerase, ATP sufturylase, luciferase, apyrase Detection - Pyrophosphate release activates luciferase, which breaks down luciferin and gives off light

What was the first living organism to have its entire genome sequenced

Haemophilus influenza

Shotgun Sequencing

Method where small DNA fragments are randomly generated from larger pieces and sequenced

What is the equation for probability in shotgun sequencing?

P = e^3m P = Probability that a base will be left unsequenced e = natural log m = number of bases sequenced / total genome size

What did the scientists do the the H. influenzae fragments after breaking them into fragments?

Randomly cloned into vectors and grown in E. coli and sequenced

High-throughput sequencing

Rapidly sequencing large amounts of DNA

genome sequencing projects

Research aimed at determining the DNA sequence of the entire genome of a species

What is the most efficient and inexpensive way to sequence genomes?

Shotgun sequencing

Parallel sequencing

Simultaneously perform many sequencing runs in multiple gel-filled capillary tubes

How was the DNA from H. influenzae broken into fragments?

Sound waves

In pyrosequencing, what does the amplitude of the light indicate?

The stronger the light, the more nucleotides were added sequentially. (2x bright means 2 of the same nucleotide added AA,GG,TT,CC)

What is metagenomics?

The study of a complex mixture of genetic material from an environmental sample

What happened to the fragments after being grown in E. coli

Underwent dideoxy sequencing

During pyrosequencing, the release of pyrophosphate after addition of a nucleotide is monitored through a series of chemical reactions. In the first reaction, pyrophosphate and adenosine 5' phosphosulfate are the substrates for ATP synthesis by the enzyme ATP sulfurylase. The ATP produced in this reaction is combined with luciferin by the enzyme ________ to produce __________.

luciferase, light

In pyrosequencing, the incorporation of a nucleotide into a growing DNA strand is monitored by measuring the release of ______.

pyrophosphate (PPi)