Biol 240: Genomics

genome

entire complement of genetic information.

Includes genes, regulatory sequences, non-coding sequences

genomics

the discipline involving mapping, sequencing, analyzing and comparing genomes

functional genomics

assigning function to unknown genes by analyzing biochemical and physiological effects of mutants

Three needs created by the development of recombinant DNA

•improved DNA sequencing techniques

•formats for storage of large data sets

•computational tools for analysis of "big data" data

Sanger dideoxy sequencing

dideoxynucleotides become part of RNA creation and stop RNA formation at specific points because of lack of OH group. Creates RNA strands of known length

Three steps of Sanger dideoxy sequencing

cloning a gene fragment of interest

DNA synthesis

gel electrophoresis

What labels do automatic methods use and why?

fluorescent lables, because standard radioactive labels are less safe and more expensive

how long are the strands, how long does it take to get them, and what quality are they?

700-1000 base pairs. get them in a few hours, very high quality with fluorescent labels

Longer sequences are obtained by _____ ______

primer walking

what is primer walking?

using repeated rounds of sequencing with primers complementary to the end of the last segment sequenced

(Design primers that can bind to end of insert, then end of sequencing to have multiple sequences)

Shotgun Sequencing

attempting to sequence the entire genome in one step

(much faster)

steps of shotgun sequencing

DNA fragments are sheared, cloned, then sequenced. After, a software aligns/assembles the full genome (need 10x coverage to do so successfully)

high-throughput sequencing

Rapid DNA sequencing on a micro scale in which many fragments of DNA are sequenced in parallel.

5 types of high-throughput sequencing:

Pyrosequencing, Ion torrent, Illumina, Pacific Bioscience, Oxford Nanopore

Pyrosequencing method

detects nucleotide additions to the end of a strand of DNA by production of light by luciferase. Short reads (~500-700 bases). (is obsolete now)

Ion Torrent method

similar to pyrosequencing but detects nucleotide additions to the end of a strand of DNA by production of light by pH change (proton released). Short reads (~400 bases)

Illumina method

is industry standard for short reads (~150-300 bases). Fluorophores added to template strands on glass array - high res fluorescence scan.

Pacific Bioscience method (PacBio)

used for long reads (>10,000 bases!), recording fluorophore incorporation in real time. High error rates.

Oxford Nanopore method

passes DNA strands through massively parallelized "nanopores", recording current disruptions that are specific to each DNA base. Long reads and relatively high error rates (>1kb to 1 mb!). uses min-ion

Bioinformatics

use of computational tools to analyze, compare, assemble, and store, DNA & protein sequences

annotation of genomes helps researchers identify ____ _______ ______

open reading frames

Open Reading Frames allow us to:

better determine the start and stop points for a given gene

Transcriptome

collection of transcribed mRNA molecules in a cell

a library of the expressed mRNA molecules of a cell can be formed as a _____ library using reverse transcriptase. Can be sequenced directly, or by microarrays

cDNA

How is RNA sequencing performed?

conversion of mRNA into cDNA by reverse transcriptase. cDNA is then sequenced using rapid automated methods

Microarrays

A method for examining transcriptional activity of all genes in a cell simultaneously. Probe DNA fragments are amplified by PCR and placed on a glass slide in a known pattern. The total cell mRNA is converted to cDNA by reverse transcriptase, labeled with a fluorescent molecule, and passed over the microarray slide. The more intensely a "spot" on the microarray lights up, the more cDNA is present

5 uses for Microarrays:

-global gene expression

-expression of specific gene classes under different conditions (regulons)

-expression of genes with unknown function

-comparison of gene content in related organisms

-species identification

Proteome

the collection of expressed proteins in a cell

4 methods used to study the proteome:

•2D-polyacrylamide gel electrophoresis (PAGE)

•mass spectrometry

•x-ray crystallography

•nuclear magnetic resonance (NMR)

2D-PAGE method:

allows for the separation of proteins on a polyachrimalide gel based on isoelectric point and mass

What can be used to determine the amino acid sequences of the polypeptides from he 2D-PAGE?

mass spectrometry

From this, how would you determine the identity of the protein?

by comparing the sequence to known protein sequences

X-ray crystallography

An X-ray beam is shot at crystallized protein and the diffraction pattern is used to discern protein shape

drawback to x-ray crystallography?

it is very hard to crystallize proteins

Nuclear Magnetic Resonance

•measures distances between atomic nuclei and can measure proteins in solution. Only measures smaller proteins (30 kDa)

Metagenomics

involves construction and analysis of gene libraries from DNA extracted directly from complex microbial communities

3 steps of metagenomics

-obtain DNA from area of interest

-sequence

-analyze (eliminate known sequences to then find new genes, potentially from new organisms)

You did it man

Good Job!!!!