runs DNA to red. Why?
Gel electrophoresis
autoradiograph
+
-
Utility
* Lacking 3 5 exonuclease
http://www.neb.com/nebecomm/tech_reference/polymerases/properties_dna_polymerases.asp
Log/Linear relationship between PCR product and cycle number
Rn
Plateau
700 nm
650nm
600nm
550nm
500nm
450nm
400nm
Cy5.5
Cy5
Flourescein Cy 3.5 TAMRA Texas Red.X Cy3 ROX SYBR Green 6-FAM ROX TAMRA HEX Cy3 Flourescein TET AmpF1 Blue TET SYBR Green 6-FAM HEX AmpF1 Blue
Texas Red.X
Cy5.5
Cy5
Cy 3.5
700 nm
650nm
600nm
550nm
500nm
450nm
400nm
0.05 ng 0.1 ng 0.5 ng 1 ng 5 ng 25 ng
negatives
log Co = 0.934 so Co = 8.6 ng
Most researchers dont quantitate the Co of their unknown samples using the standard curve method. Instead, they prefer mathematical methods such as the: Pfaffl Method. (M.W. Pfaffl. 2001. A new mathematical model for relative quantification in real-time RT-PCR Nucleic Acids Res. 2001, 29(9):e45) which is based of efficiency curves and Ct Method, a approximation method based on the difference in the threshold cycle normalized to the endogenous control gene and relative to the calibrator (i.e., your control sample).
2002 JAIC/Davis Bioscience Fund
March 15, 2010
DNA sequence from automatic sequencer (Applied Biosystems)
Next Generation Sequencing in Genomics
Roche 454: Pyrosequencing
Prepare Adapter Ligated ssDNA Library Bead loading
Clonal amplification on beads
Load beads & enzymes in PicoTiterPlateTM Plate is loaded into 454 Sequencer
DNA capture Bead containing ~107 copies of a single clonal ssDNA
A
A
T
C
G
G
C
A
T
G
C
T
A
A
A
A
G
T
APS Sequencing by sequential nucleotide flow, primer extension & pyrophosphate signal generation Sulfurase Luciferase ATP
PP T i
C
A
Anneal Primer
luciferin www.454.com
Light oxy + luciferin
Roche 454: Pyrosequencing
Uses the pyrophosphate to synthesize ATP and generate light with luciferase One of the 4 dNTPs is added at a time Excess nucleotides are degraded after each addition Strings of repeated bases are read as higher signal peaks, but the relative increase is not linear with the length of the homopolymer run.
Roche 454: Pyrosequencing: Paired-end reads
Shearedand adaptedDNA
Necessary for plant genomes to span repeats ~400 bp read length; up to 20 kb paired-end reads.
Shearand isolateadaptor containing fragments AddA/B adaptorsand amplify Circularize (usescrelox)
Illumina (Solexa) Genome Sequence Analyzer (GSA)
PCR-based cluster formation on slide; Sequencing by synthesis: similar concept to Sanger (fluorescent, 3-blocked nucleotides) Scan slide after each synthesis cycle
Illumina Method
A. B.
Adapter DNA fragment Dense primer lawn Adapter
Sample Prep: Ligate adapters to ends of random primed cDNA
Random binding of single stranded cDNA to primers Fragments become double stranded Attached Free terminus
C.
Bridge Amplification with unlabeled dNTPs
D.
E.
Denature double stranded molecules Attached
F.
35 cycles of bridge amplification Cluster
G.
H.
1st SBS Cycle with 4 cleavable Image capture & dye terminators Centerase Genome Resources 2009 B for calling National
How much sequence can you get?*
Method 454-flex 454-titanium
# of Reads ~ 400,000 1.0 1.2 million
Read length ~ 250 bp ~ 400 bp 50 100 bp 25-50 bp
Total sequence ~ 100 Mb Up to 500 Mb 10-15 Gb (x2) 15 Gb (x2)
Illumina/Solexa 10-15 million per channel (x7) ABI SOLiD 300 million per slide
Unit costs for 454, Solexa and SOLiD are comparable, although paired ends and longer reads do make Solexa more expensive at the upper end.