Nucleic Acid Tracing
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.
Background & General Information
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/Bioinformatics/Projects/hint2.htm
www.dur.ac.uk/stat.web/Bioinformatics/DNA_corner.htm
http://www.dur.ac.uk/biological.sciences/Bioinformatics/DNA_c
orner.htm
bio.fsu.edu/~stevet/BioInfoSurvey.BCH5425.05.ppt#256,1,The
“Nuts and Bolts” of ‘doing’ bioinformatics with the Wisconsin
Package at FSU
Nick-translation Labeling
Nick-translation defined:
http://www.fmv.ulg.ac.be/
genmol/MODGEN/Chapte
rIII/SR5_2A.htm
DNA labeling & other NA protocols:
http://info.med.yale.edu/genetics/ward/tavi/n_label.html
Random Priming Labelling
http://www.bio.davidso
n.edu/courses/genomi
cs/method/randomprim
ing.html
http://www.chups.jussieu.fr/polys/bioc
himie/BGbioch/POLY.Chp.9.12.html
Polymerase
Chain
Reaction
One of the most
common means of
amplifying, modifying,
or tagging nuclei acids.
Amplification may
reach millions of copies
after 20-30 cycles.
PCR Parameters
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
Variants of PCR
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)
Landers et al. at
Virginia are working to
put PCR in a microfluidics system that
uses IR heating.
http://www.faculty.virgi
nia.edu/landers/Image
s/pcr2.gif
Real Time PCR
http://www.promega.co
m/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/TechUpdate
s.htm
Real Time sites:
http://dna-9.int-med.uiowa.edu/realtime.htm
Taqman Approach to PCR
Perkin Elmer Taqman PCR:
http://cgr.otago.ac.nz/SLIDES/TAQMAN/INDEX.HTM
Reverse Transcriptase PCR
http://www.promega.com/guides/
http://www.westburg
.eu/en/site/lifesciences/pcr-rt-pcr
Rolling Circle Amplification
http://courses.agri.hu
ji.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/Seminars_
and_news
http://www1.gelifescienc
es.com/APTRIX/upp0107
7.nsf/Content/sample_pr
eparation~product_select
ion_category~rolling_circ
le_amplification/$file/flas
h.htm
LCR
T4 Ligase:
http://dwb.unl.edu/Teacher/NSF/C08/C08Links/www.worthi
ngton-biochem.com/manual/D/DNAT4L.html
http://www.biochem.uwo.ca/community/molbio/ligate.html
Southern Protocol plus: Southern & Northern Blots
http://www.ableweb.org/volumes/vol-12/1-karche/1karche.htm
http://www.healthsystem.virginia.edu/internet/transgenicmouse/southern.cfm
http://center.intron.co.kr/m
ethod/protocol_2.asp
http://www.bio.davidson.edu
/courses/genomics/method/
Northernblot.html
TUNEL Assays
Detection of apoptotic
cells in the mouse testis
using the TUNEL assay
(methyl green
counterstaining).
http://sciencepark.mdanderson.or
g/fcores/histology/histology_pict
s/staining/apoptosis_assays/Tune
lTesticle_10xMethgre-04.jpg
Apoptosis Assays:
http://sciencepark.mdanderson.org/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.html
Protocols:
www.animal.ufl.edu/hansen/protocols/tunel.htm
http://sciencepark.mdanderson.org/flow/files/TUNEL.html
DNA
Microarray
Tutorials:
http://www.trans
criptome.ens.fr/s
gdb/presentation
/principle.php
http://www.ebi.a
c.uk/ebisearch/
search.ebi?db=
allebi&query=M
icroarray
Point mutation detection with DNA microchips:
http://www.clinchem.org/cgi/reprint/46/10/1555.pdf
Reagents & methods:
www.cambridgebluegnome.com/Products/
www.bioxing.com/Products/bxdesigner.htm
Statistical methods:
http://www.garnetthenley.com/Descriptive.pdf
DNA Sequencing
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; 100’s of bp/day; low # bp/read;
>>$250/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
Maxam-Gilbert Sequencing
http://www.campus.skelleftea.se/
biomine/molecular/index_14.htm
Sanger Sequencing
http://www3.appliedbiosystems.com/AB_Home/app
licationstechnologies/DNASequencingbyCapillaryE
lectrophoresis/index.htm
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 α # N’s added
100 Mb/run (7h); 250 bp/read; $84/Mb
http://www.454.com/enabling-technology/the-technology.asp
Genome Sequencer 20 System 2006 Roche Diagnostics GmbH.
Genome Sequencer 20 System 2006 Roche Diagnostics GmbH.
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?ID=203
http://seqanswers.com/forums/showthread.php?t=21
Illumina
Steps 1-6
http://seqa
nswers.co
m/forums/
showthrea
d.php?t=21
Illumina
Steps 7-12
http://seqa
nswers.co
m/forums/
showthrea
d.php?t=21
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’ N’s, 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/Product/Solid
_Knowledge/flash/102207/solid.html
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); > 25 b/read; $2.40/MB
http://www.helicosbio.com/Technology/tabid/62/Default.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?q=technologyintroduction
Nanopore Sequencing
Detection of exonuclease product dNDPs via modified
electrical flow in a nanopore tethered to the enzyme
http://www.nanoporetech.com/sequences
http://www.pacificbiosciences.com/template/image_popup.php?
img=tech/zmw_dna_poly_phos_nucl_lg.jpg&imgTitle=ZMW%20
with%20DNA%20polymerase%20and%20phospholink%20nucle
otides&background=#000
Background:
http://www.roswellpark.org/document_3636_639.html
Methods:
http://www.mcb.uct.ac.za/Sequencing%20Service%20web/
index.htm
http://www.epibio.com/litindex.asp?method=alpha
New Sequencing Approaches
454 Life Sciences, Pyrosequencing
Original Description
https://www.roche-appliedscience.com/servlet/RCConfigureUser?URL=StoreFramesetView&st
oreId=10202&catalogId=10202&langId=-1&countryId=us
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
Additional Molecular Methods (especially Drosophila):
http://www.dhgp.org/current/index.html
More Methods Manuals:
http://www.dwalab.ca/labman/index.html
http://www.uhmc.sunysb.edu/bioscience/methods/m
ethods.htm
http://www.methods.info/