Class Notes (837,550)
Canada (510,314)
Biology (1,303)
BIOL 205 (111)
Prof. (9)
Lecture 13

Lecture 13.pdf

48 Pages
Unlock Document

BIOL 205

Next generation sequencing methods- Pyrosequencing 1) Add a single DNA to a single bead! 2) Place bead in one of 400,000 picoliter wells 3) PCR amplify DNA synthesis 4) Uses luminescence to detect sequence Sequences 400 Mb in 4 hrs Principle of Pyrosequencing Pyrosequencing technology, which is based on the principle of sequencing by synthesis. Step 1 A sequencing primer is hybridized to a single-strandedPCR ampliconthat serves as a template,and incubatedwith the enzymes,DNA polymerase,ATP sulfurylase,luciferase, and apyrase as well as the substrateadenosine 5' phosphosulfate(APS), and luciferin. Step 2 The first deoxribonucleotide triphosphate(dNTP) is added to the reaction.DNA polymerasecatalyzesthe incorporationof the deoxyribo-nucleotidetriphosphateinto the DNA strand, if it is complementaryto the base in the templatestrand. Each incorporation event is accompaniedby release of pyrophosphate(PPi) in a quantity equimolar to the amountof incorporatednucleotide. Step 3 ATP sulfurylase converts PPi to ATP in the presence of adenosine5' phosphosulfate(APS). This ATP drives the luciferase-mediatedconversionof luciferin to oxyluciferin that generates visible light in amountsthat are proportionalto the amountof ATP. The light produced in the luciferase-catalyzedreactionis detectedby a charge coupled device (CCD) camera and seen as a peak in the raw data output(Pyrogram).The height of each peak (light signal) is proportionalto the number of nucleotidesincorporated. Step 4 Apyrase, a nucleotide-degradingenzyme, continuouslydegrades unincorporatednucleotidesand ATP. When degradationis complete,another nucleotide is added. Step 5 Additionof dNTPs is performed sequentially.It should be notedthat deoxyadenosine alfa-thiotriphosphate(dATPαS) is used as a substitutefor the natural deoxyadenosinetriphosphate(dATP) since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. As the process continues,the complementaryDNA strand is built up and the nucleotidesequence is determinedfrom the signal peaks in the Pyrogramtrace. Illumina/Solexa sequencing -Latest innovation Fluorescence is measured as DNA synthesis takes place on millions of spots simultaneously Key is fluorescent reversible Terminator nucleotides (different wavelength for each! -incorporate at complementary Nucleotide –terminates chain & fluoresces. -Fluorescent dye and chain terminator removed –incorporated nucleotide serves as primer for next incoming TRANSCRIPTION –Chapter 8 The four ribonucleotides found in RNA Figure 8-2 Opposite DNA strands can serve as template for RNA Overview of transcription Figure 8-4 Many RNAs can be simultaneously transcribed from a gene Figure 8-5 Promoter sequences in E. coli 5’ Untranslated Region 3’UTR +1 is the start of Transcription Transcription initiation in prokaryotes No primer necessary β subunit has catalytic activity α and ω involved in assembly β’ binds to DNA ϭ binds to -10 to -35 RNA polymerase holoenzyme positions holoenzyme Α2ββ’ω -role in melting DNA around -10 -dissociates as transcription begins Elongation and termination of transcription NTP + (NMP) n (NMP) +nPPi Elongation and termination of transcription Terminator sequence ~ 40 bp in 3’UTR ending in GC –rich stretch followed by string of 6 or more A’s - creates a transcript that forms a hairpin -Causes polymerase to pause, backtrack encounters hairpin and disassembles Second termination ρ = Rho protein mechanism in prokaryotes Prokaryotic and eukaryotic transcription and translation compared Compartmentalization! Eukaryotes have 3 RNA polymerases • RNA polymerase I –localized in the nucleolus -transcribes ribosomal RNA 28S, 18S and 5.8S rRNA – -component of ribosomal subunits • RNA polymerase II – transcribes all mRNA • RNA polymerase III – transcribes tRNA, 5S rRNA RNAPOLYMERASE II TRANSCRIBED GENES most cellular mRNAs 5’Flanking region Promoter-proximal elements or cis-acting control elements or upstream activator sequences (UAS) DNA Enhancer ~-100 TATA Element BOX > +/-1000 -30 Control elements : Different in almost every gene Each recognized by protein factors known as Transcription factors Overview of transcriptional regulation Figure 11-2 Modified histone tails protrude from the nucleosome Figure 11-12 Histone Acetylated histones acetyltransferase associated with (HAT) actively transcribed transfer
More Less

Related notes for BIOL 205

Log In


Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.