As DNA

1
As DNA RNA are polymers comprised of a very
limited number of distinct monomers, biomedical
tracing of these molecules principally consists
of introducing modified base monomers into the
structure of the nucleic acid of interest.
Modification may also occur by adding molecular
marker groups with chemistries similar to what we
have described for proteins other molecules.
Radioactivity, chromogens, fluorogens, particles,
density labels have all been used to tag
nucleic acids. Antibodies also exist that can
distinguish the 2D 3D features of specific
nucleic acids or sequences.
2
Background http//www.gene-quantification.info/
NA applets http//www.oligo.net/
Enzymology of DNA replication Nucleic Acid
Labeling\CUHKBio2310TutorialFile1Expt3NABasics.pdf
www.ufrgs.br/depbiot/blaber/section1/section1.htm
Biochemistry of polymerases ligases for NA
manipulation www.chups.jussieu.fr/polys/biochimie
/BGbioch/POLY.Part.I.html
Bioinformatics Info www.dur.ac.uk/stat.web/Bioin
formatics/Projects/hint2.htm www.dur.ac.uk/stat.we
b/Bioinformatics/DNA_corner.htm http//www.dur.ac.
uk/biological.sciences/Bioinformatics/DNA_corner.h
tm
bio.fsu.edu/stevet/BioInfoSurvey.BCH5425.05.ppt2
56,1,The Nuts and Bolts of doing
bioinformatics with the Wisconsin Package at FSU
3
Nick-translation defined http//www.fmv.ulg.ac.be
/genmol/MODGEN/ChapterIII/SR5_2A.htm
DNA labeling other NA protocols http//info.med
.yale.edu/genetics/ward/tavi/n_label.html
4
http//www.bio.davidson.edu/courses/genomics/metho
d/randompriming.html
http//www.chups.jussieu.fr/polys/biochimie/BGbioc
h/POLY.Chp.9.12.html
5
One of the most common means of amplifying,
modifying, or tagging nuclei acids.
Amplification may reach millions of copies after
20-30 cycles.
6
NA length, redundancy, GC/AT content Polymerase
features (Taq, Vent, ) Mg Primers dNTPs,
NTPs Time temperature of denaturation Time
temperature of primer annealling Time
temperature of extension Presence of additives
(intended, impurities) Number of cycles Nature of
labeled dNTPs, NTPs
7
RT-PCR (Reverse Transcriptase-PCR) RT-PCR
(Real-Time PCR) using hairpin primers using
intercalation labels Universal primers Rolling
circle amplification LCR (Ligase Chain Reaction)
8
Landers et al. at Virginia are working to put PCR
in a micro-fluidics system that uses IR heating.
http//www.faculty.virginia.edu/landers/Images/pcr
2.gif
9
http//www.promega.com/paguide/paguide_us.pdf
(Also includes many, many more protocols 299 p,
24MB)
Methods notes that include competitive
quantitative PCR http//ccm.ucdavis.edu/cpl/Tech
20updates/TechUpdates.htm
Real Time sites http//dna-9.int-med.uiowa.edu/r
ealtime.htm
10
Perkin Elmer Taqman PCR http//cgr.otago.ac.nz/S
LIDES/TAQMAN/INDEX.HTM
11
http//www.promega.com/guides/
http//www.westburg.eu/en/site/life-sciences/pcr-r
t-pcr
12
http//courses.agri.huji.ac.il/71953/ic7.htm
Alia L. Merla, Amersham presented an alternative
to DNA plasmid amplification, 7/16/03, Seminar
at UCLA, http//genoseq.ucla.edu/action/view/Semin
ars_and_news
http//www1.gelifesciences.com/APTRIX/upp01077.nsf
/Content/sample_preparationproduct_selection_cate
goryrolling_circle_amplification/file/flash.htm
13
T4 Ligase http//dwb.unl.edu/Teacher/NSF/C08/C08L
inks/www.worthington-biochem.com/manual/D/DNAT4L.h
tml http//www.biochem.uwo.ca/community/molbio/lig
ate.html
14
Southern Protocol plus http//www.ableweb.org/vol
umes/vol-12/1-karche/1-karche.htm http//www.healt
hsystem.virginia.edu/internet/transgenic-mouse/sou
thern.cfm
http//center.intron.co.kr/method/protocol_2.asp
http//www.bio.davidson.edu/courses/genomics/metho
d/Northernblot.html
15
Detection of apoptotic cells in the mouse testis
using the TUNEL assay (methyl green
counterstaining). http//sciencepark.mdanderson.or
g/fcores/histology/histology_picts/staining/apopto
sis_assays/TunelTesticle_10xMethgre-04.jpg
Apoptosis Assays http//sciencepark.mdanderson.or
g/fcores/histology/apop.html http//www.compucyte.
com/apoptosis.htm http//www.ikp.unibe.ch/lab2/REG
/index.htm http//www.phnxflow.com/apo.overview.ht
ml
Protocols www.animal.ufl.edu/hansen/protocols/tun
el.htm http//sciencepark.mdanderson.org/flow/file
s/TUNEL.html
16
Tutorials http//www.transcriptome.ens.fr/sgdb/pr
esentation/principle.php http//www.ebi.ac.uk/ebis
earch/search.ebi?dballebiqueryMicroarray
Point mutation detection with DNA
microchips http//www.clinchem.org/cgi/reprint/46
/10/1555.pdf
Reagents methods www.cambridgebluegnome.com/Pro
ducts/ www.bioxing.com/Products/bxdesigner.htm
Statistical methods http//www.garnetthenley.com/
Descriptive.pdf
17

• Maxam-Gilbert
• Amplify NA or fragments using vectors or PCR
• Radiolabel 5 end
• Fragment DNA using base selective chemical
  agents
• Separate products on PAGE or capillary
  electrophoresis
• Read autoradiograms
• Manual protocol 100s of bp/day low bp/read
  gtgt250/Mb
• Sanger
• Amplify NA or fragments using vectors or PCR
• Radio- or dye-labelled ddNTPs polymerase used
  in a final
• labeling replication/amplification
• Separate products on PAGE or capillary
  electrophoresis
• Read autoradiograms or laser excited
  electroperograms
• produced by PMT or CCD
• Manual, Fast ABI sequencer kb/day 400-800
  bp/read 234/Mb

18
Maxam-Gilbert Sequencing
http//www.campus.skelleftea.se/biomine/molecular/
index_14.htm
Sanger Sequencing
19
http//www3.appliedbiosystems.com/AB_Home/applicat
ionstechnologies/DNASequencingbyCapillaryElectroph
oresis/index.htm
20

• Next/Second Generation
• Pyrosequencing 454 Life Sciences
• Emulsion PCR of NA fragments
• Picotiter well serial reaction with dNTPs
• Luciferase coupling of luciferin to ATP from PPi
  via
• sulfurylase exchange of S on APS
• CCD detection of light pulse amplitude a Ns
  added
• 100 Mb/run (7h) 250 bp/read 84/Mb

http//www.454.com/enabling-technology/the-technol
ogy.asp
21
Genome Sequencer 20 System
2006 Roche Diagnostics GmbH.
22
Genome Sequencer 20 System
2006 Roche Diagnostics GmbH.
23
Next/Second Generation (cont.)

• Bridge-amplification fluorescent dyes
  Illumina
• Adapter ligation, surface attachment, bridge
  amplification
• Denature, cluster
• Single F-dNTP extension
• Image clusters
• Deblock remove fluorophore
• Repeat labeling detection
• 1300 Mb/run (4d) 32 - 40 bp/read 6/Mb

http//www.illumina.com/pages.ilmn?ID203
http//seqanswers.com/forums/showthread.php?t21
24
Illumina Steps 1-6
http//seqanswers.com/forums/showthread.php?t21
25
Illumina Steps 7-12
http//seqanswers.com/forums/showthread.php?t21
26

• Next/Second Generation (cont.)
• Ligation-based, dual nucleotide SOLiD
• Ligate adapters hybridize to beads w/
  complementary adapters
• Amplify w/ emulsion PCR
• Tether beads to a surface
• Hybridize anchoring adapters 1st of
  F-8mer-known-diN probes
• Ligate probes, wash, read signal
• Remove F- 3 3 Ns, wash
• Repeat hybridization, ligation reading
• Software calls sequences from color patterns
• 3000 Mb/run (5d) 25 - 35 bp/read 6/Mb

http//marketing.appliedbiosystems.com/images/Prod
uct/Solid_Knowledge/flash/102207/solid.html
27
Third Generation Methods
Helicos Technique appears similar to Illumina but
is done on single DNA strands w/o prior
amplification 7.5 GB/run (14d) gt 25 b/read
2.40/MB
http//www.helicosbio.com/Technology/tabid/62/Defa
ult.aspx
Single Molecule Real Time Tethered polymerase
adds F-dNTP then cleaves F to add next F-dNTP
CCD images single polymerase rxns
http//www.pacificbiosciences.com/index.php?qtech
nology-introduction
Nanopore Sequencing Detection of exonuclease
product dNDPs via modified electrical flow in a
nanopore tethered to the enzyme
http//www.nanoporetech.com/sequences
28
http//www.pacificbiosciences.com/template/image_p
opup.php?imgtech/zmw_dna_poly_phos_nucl_lg.jpgim
gTitleZMW20with20DNA20polymerase20and20phosp
holink20nucleotidesbackground000
29
Background http//www.roswellpark.org/document_36
36_639.html
Methods http//www.mcb.uct.ac.za/Sequencing20Ser
vice20web/index.htm http//www.epibio.com/litinde
x.asp?methodalpha
30
New Sequencing Approaches
454 Life Sciences, Pyrosequencing
Original Description
https//www.roche-applied-science.com/servlet/RCCo
nfigureUser?URLStoreFramesetViewstoreId10202ca
talogId10202langId-1countryIdus
http//www.liv.ac.uk/agf/454sequencing.html
http//www.dkfz.de/gpcf/242.html
http//jeb.biologists.org/cgi/content-nw/full/210/
9/1518/FIG2
http//www.genengnews.com/sequencing/supp_02.aspx
31
(No Transcript)
32
Additional Molecular Methods (especially
Drosophila) http//www.dhgp.org/current/index.htm
l
More Methods Manuals http//www.dwalab.ca/labman/
index.html http//www.uhmc.sunysb.edu/bioscience/m
ethods/methods.htm http//www.methods.info/