Diseo de primers

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Diseño de primers
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Francis Crick
Alex Rich Leslie Orgel James
Watson
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Escenarios frecuentes en el diseño de primers

• Secuencia de DNA conocida
• Diagnóstico, genotipificación
• Tener cuidado si es DNA o cDNA
• Secuencia de DNA desconocida
• Se busca secuenciar el gen en cuestión
• Regiones conservadas en genes homólogos de
  organismos cercanos
• Extremos amino ó carboxilo terminal de la
  proteína codificada es conocida

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Secuencia de DNA conocida

• Los primers se diseñan a partir de criterios de
  optimalidad
• Estabilidad térmica del complejo primer-DNA
• Estructura secundaria
• Dímeros
• Temperatura media de fusión
• Especificidad

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PCR
1 copy
and - strands of template
forward primer
reverse primer
amplified DNA
5
3
3
5
2 copies
5
3
5
3
4 copies
etc.
8 copies
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Ciclos deTemperatura

• Temperatura de hibridación (annealing) (usual
  45-60?C) allows primers to hybridize to template
• Temperatura de extensión (usually 72?C) allows
  polymerase to extend starting at the primer
• Temperatura de denaturación (usually 95?C)
  separates strands

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• Tiempo de vida media de Taq polymerasa
• aprox. 30 min a 95C,
• Es lar razón por la que después de 30 ciclos de
  amplificación se pierde eficiencia
• Es posible reducir la temperatura de
  denaturación luego de 10 ciclos de amplificación
  ya que la longitud media del DNA blanco se ha
  reducido cercanamente al tamaño del amplicón
  para templados de 300bp o menos, la temperatura
  de denaturación puede reducirse a valores tan
  bajos como 88C para un 50 de (GC) (Yap and
  McGee, 1991), por lo que es posible llegar hasta
  40 ciclos sin disminuir sustancialmente la
  eficiencia de la enzima.

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Durante el PCR se da una liberación de
pirofosfato que juntamente con el producto van a
comportarse de manera inhibitoria
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Considerations About Primers
Characteristics of primers
Thoughts on primer design
Uniqueness
Specificity Specific for the intended target
sequence (avoid nonspecific hybridization)
Length
Base Composition
Internal Stability
Stability Form stable duplex with template under
PCR conditions
Melting Temperature
Annealing Temperature
Internal Structure
Compatibility Primers used as a pair shall work
under the same PCR condition
Primer Pair Matching
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Uniqueness
There shall be one and only one target site in
the template DNA where the primer binds, which
means the primer sequence shall be unique in the
template DNA. There shall be no annealing site in
possible contaminant sources, such as human, rat,
mouse, etc. (BLAST search against corresponding
genome)
Template DNA 5...TCAACTTAGCATGATCGGGTA...GTAGCAG
TTGACTGTACAACTCAGCAA...3
TGCTAAGTTG
CAGTCAACTGCTAC
NOT UNIQUE!
Primer candidate 1 5-TGCTAAGTTG-3
UNIQUE!
Primer candidate 2 5-CAGTCAACTGCTAC-3
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Length
Primer length has effects on uniqueness and
melting/annealing temperature. Roughly speaking,
the longer the primer, the more chance that its
unique the longer the primer, the higher
melting/annealing temperature. Generally
speaking, the length of primer has to be at least
15 bases to ensure uniqueness. Usually, we pick
primers of 17-28 bases long. This range varies
based on if you can find unique primers with
appropriate annealing temperature within this
range.
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Base Composition

• Base composition affects hybridization
  specificity, melting/annealing temperature and
  internal stability.
• Random base composition is preferred. We shall
  avoid long (AT) and (GC) rich region if
  possible.

Template DNA 5...TCAACTTAGCATGATCGGGCA...AAGATGC
ACGGGCCTGTACACAA...3
TGCCCGATCATGCT

• Usually, average (GC) content around 50-60
  will give us the right melting/annealing
  temperature for ordinary PCR reactions, and will
  give appropriate hybridization stability.
  However, melting/annealing temperature and
  hybridization stability are affected by other
  factors, which well discuss later. Therefore,
  (GC) content is allowed to change.

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Thermodynamics

• Gibbs Free Energy, G
• Describes the energetics of biomolecules in
  aqueous solution. The change in free energy, ?G,
  for a chemical process, such as nucleic acid
  folding, can be used to determine the direction
  of the process
• ?G0 equilibrium
• ?Ggt0 unfavorable process
• ?Glt0 favorable process
• Thus the natural tendency for biomolecules in
  solution is to minimize free energy of the entire
  system (biomolecules solvent).

•  ?G ?H - T?S
• ?H is enthalpy, ?S is entropy, and T is the
  temperature in Kelvin.
• Molecular interactions, such as hydrogen bonds,
  van der Waals and electrostatic interactions
  contribute to the ?H term. ?S describes the
  change of order of the system.
• Thus, both molecular interactions as well as the
  order of the system determine the direction of a
  chemical process.
• For any nucleic acid solution, it is extremely
  difficult to calculate the free energy from first
  principle
• Biophysical methods can be used to measure free
  energy changes

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Internal Stability
Stability Profile Internal stability is
calculated with entropy values of neighbor
nucleotides. Usually, We draw a graph of ?G for
all nucleotides of the primers. This is known as
the stability profile.
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3 Stability 5 Stability
Primer elongation starts at 3 end. Therefore, as
long as 3 end hybridizes to the template stably,
the elongation begins. 5 end sequence plays less
important role. This feature can be utilized for
modifying 5 end so that there will be a
restriction enzyme site. However, this feature
also brings out a problem if 3end of the
primer has 3 or more than 3 C/G, it can almost
bind stably to any site where there are 3
complement G/C bases.
Template DNA 5...TCAACTTAGCGTGATCGATTA...AAGATTC
GCGTTAGCTGTACACAA...3
5-AATCGATCTCGC-3
Ideal situation will be stable 5-termini less
stable 3-termini, which eliminates false priming
due to annealing of 3'-half of primer only. We
prefer the 5 end has 1 or two G/C bases (GC
clamp) and the 3 end has no more than 1 G/C base.
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Melting Temperature
Melting Temperature, Tm the temperature at
which half the DNA strands are single stranded
and half are double-stranded.. Tm is
characteristics of the DNA composition Higher
GC content DNA has a higher Tm due to more H
bonds. Calculation Method 1 (Base Composition)
Tm ( A T ) ? 2?C ( C G ) ? 4?C
(lt20bp) Method 2 (Salt Adjusted) Used by GCG
and Primer3 Tm 81.5 16.6 ? log10Na
0.41 ? GC 0.65 ? formamide 675/length
mismatch Method 3 (Nearest Neighbor) Used by
OLIGO ?H / (10.8 ?S R ? ln (c / 4))
273.15 16.6(log10K) where ?H is the sum of
enthalpy of the nearest neighbors, ?S is the sum
of entropy of the nearest neighbors, c is the
molar concentration of primer, and R is the gas
constant (1.987). As you can tell from the
equation higher GC, higher Tm Higher probe,
higher Tm higher K, higher Tm Differences are
sometimes significant, like 8 degrees, and
sometimes trivial, like 0.1 degree. Try different
software to calculate Tm. Pick the common value.
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Annealing Temperature
Annealing Temperature, Tanneal the temperature
at which primers anneal to the template DNA. It
can be calculated from Tm .
Tanneal Tm_primer 4?C or 0.3 ? Tm_primer
0.7 ? Tm_product 14.9 Where Tm_primer is the
melting temperature for primer and Tm_product is
the melting temperature for product.
To ensure that primers anneal to the template
before the two strands of template anneal to each
other, it required that the Tm_product Tanneal
30 ?C
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Stringency in Primer Annealing

• Stringency determines the specificity of the
  amplified DNA product. Tanneal is the most
  significant factor affecting the stringency in
  primer annealing.
• Tanneal too low ? less stringent ? primer
  matches elsewhere
• too high ? more stringent ? primer
  may fail to match
• Other factors
• GC GC pairs are more stringent than AT paris
• Salt Buffer

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Internal Structure
If primers can anneal to themselves, or anneal to
each other rather than anneal to the template,
the PCR efficiency will be decreased
dramatically. They shall be avoided.
However, sometimes these 2? structures are
harmless when the annealing temperature does not
allow them to take form. For example, some dimers
or hairpins form at 30 ?C while during PCR cycle,
the lowest temperature only drops to 60 ?C.
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Primer Pair Matching
Primers work in pairs forward primer and
reverse primer. Since they are used in the same
PCR reaction, it shall be ensured that the PCR
condition is suitable for both of them. One
critical feature is their annealing temperatures,
which shall be compatible with each other. The
maximum difference allowed is 3 ?C. The closer
their Tanneal are, the better.
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Calculation procedure for extinction (absorption)
coefficient of DNA Extinction coefficient at 260
nm, 25 degrees of Celsius, and neutral pH for the
single-strand DNA is determined by the
nearest-neighbor method
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The following table contains extinction
coefficients l/(mmol.cm)

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Summary when is a primer a primer?
5
3
5
3
5
3
3
5
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Summary Primer Design Criteria

• Uniqueness ensure correct priming site
• Length 17-28 bases.This range varies
• Base composition average (GC) content around
  50-60 avoid long (AT) and (GC) rich region
  if possible
• Optimize base pairing its critical that the
  stability at 5 end be high and the stability at
  3 end be relatively low to minimize false
  priming.
• Melting Tms between 55-80 ?C are preferred
• Assure that primers at a set have annealing Tm
  within 2 3 ?C of each other.
• Minimize internal secondary structure hairpins
  and dimmers shall be avoided.

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Computer-Aided Primer Design
Primer design is an art when done by human
beings, and a far better done by machines. 

• Some primer design programs we use
• Oligo Life Science Software, standalone
  application
• - GCG Accelrys, ICBR maintains the server.
• Primer3 MIT, standalone / web application
• http//www-genome.wi.mit.edu/cgi-bin/primer/pri
  mer3_www.cgi
• BioTools BioTools, Inc. ICBR distributes the
  license.
• Others GeneFisher, Primer!, Web Primer, NBI
  oligo program, etc.

Melting temperature calculation software -
BioMath http//www.promega.com/biomath/calc11.htm

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Primer Design on the Web

• There are a bunch of good PCR primer design
  programs on the web
• Primer 3 at the MIT Whitehead Institute
• http//www.genome.wi.mit.edu/cgi-bin/primer/primer
  3_www.cgi
• Cassandra at the Univ. of Southern California
• http//www-hto.usc.edu/software/procrustes/cassand
  ra/cass_frm.html
• GeneFisher by Folker Meyer Chris Schleiermacher
  at Bielefeld University, Germany
• http//bibiserv.TechFak.Uni-Bielefeld.DE/genefishe
  r/
• Xprimer at the Virtual Genome Center, Univ.
  Minnesota Medical School
• http//alces.med.umn.edu/rawprimer.html

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Secuencia de DNA desconocida

• Regiones conservadas en genes homólogos de
  organismos cercanos

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• gtgi175637971-1014 Caenorhabditis elegans
  essential CathePsin L (38.1 kD) (cpl-1), mRNA
• ATGAACCGATTCATTCTTCTGGCACTGGTTGCCGCCGTCGTCGCCGTCAA
  TTCGGCCAAGCTGTCCCGTC
• AAATCGAGTCGGCCATCGAGAAATGGGACGACTATAAGGAGGACTTTGAT
  AAGGAGTACTCGGAGAGCGA
• GGAGCAGACCTATATGGAGGCATTTGTCAAGAATATGATCCATATTGAGA
  ATCACAACAGAGATCACCGA
• CTCGGAAGAAAGACATTCGAGATGGGATTGAATCATATTGCTGACTTGCC
  ATTCAGCCAATACCGCAAAC
• TCAACGGTTACAGACGTCTCTTCGGTGACTCCAGAATCAAGAACTCCTCC
  TCTTTCTTGGCTCCATTCAA
• TGTTCAGGTCCCAGATGAGGTTGACTGGCGTGATACCCACCTCGTCACTG
  ATGTCAAGAACCAAGGAATG
• TGCGGATCGTGCTGGGCCTTCTCCGCCACCGGAGCCCTCGAAGGACAACA
  CGCTCGCAAGCTGGGACAAC
• TCGTCTCCCTTTCCGAGCAAAACCTCGTCGACTGCTCTACCAAGTACGGA
  AACCACGGATGCAACGGAGG
• ACTCATGGATCAAGCTTTCGAGTACATTCGTGACAACCATGGTGTCGACA
  CCGAGGAGTCATACCCATAC
• AAGGGACGTGACATGAAGTGTCACTTCAACAAGAAGACCGTCGGAGCTGA
  TGATAAGGGATACGTTGACA
• CCCCAGAAGGAGATGAGGAGCAACTTAAGATCGCTGTCGCCACCCAAGGA
  CCAATCTCTATTGCTATCGA
• CGCCGGACACCGCAGCTTCCAACTTTACAAGAAGGGAGTCTACTACGATG
  AGGAATGCTCATCCGAAGAG
• CTCGACCACGGAGTGCTTCTCGTCGGATACGGAACCGACCCAGAGCACGG
  AGACTACTGGATTGTCAAGA
• ACTCGTGGGGAGCTGGATGGGGAGAGAAGGGATACATCCGTATCGCCCGT
  AACCGCAACAATCACTGCGG
• AGTCGCCACCAAGGCCAGTTATCCATTGGTCTAA

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• gtgi2518814970-1131 Brugia malayi mRNA for
  cathepsin L-like cysteine proteinase, complete
  cds
• ATGAAGGCGTTTTTTATTCTACATTTAGCCAGTTTTCTCTTGCTTACTTA
  TGCAAATCCACTTAATGAAC
• TGGATAATGATGACACACCAGGTAAAATTCATACCTTGTATCAGCAGCAT
  TATTCGAAGTATAAAACATA
• TCTGAAAAAAATGGGCAAAAAACATGATCCATCTGTTCCGGAACCAATTC
  GATTACTTAAATTTGTACAA
• TCTTTGAAAATGATTGATGAACATAACCAGCGTTACAGTAAAGGATTGGA
  AACATACAAAGTAGATCTGA
• ACAAAATGAGCGATTGGACCGAAGAAGAGAAAGAAAGACTTCGGGGATAT
  TATCCAAATTTGACTGAATA
• TGCGGAAGGAGATTTAAGTAGAATAATCCGAGGAAATATAACAACAACAA
  TACCAAAGTCTTTTGATTAT
• AGAAAGAAAATAACTGTACTACCAGCATCAGATCAAGGTCGTTGTGGTGT
  GTGTTTTATTTTCTCAGCAT
• TAGGAGCGCTTGAAATGTATGTGGCGCTCAGGACTAAAAAACGGCCGGTA
  AAACTATCAGTACAGGATGT
• AATGGATTGTAGTGGAATGGAAAAATGCAAAGGAAGAGGCGGAAATGAAC
  CTGCGGTTTTTCGTTGGGTT
• GCTGAGCACGGTGTAAAGACCGATAAGAGTTACCCATATAAGGAAAATGA
  TAGTGTTTCATGCCCGAGAA
• ATACCCCACAACGACGAAAGTATGGTTTAGCTGATGCATTCTATTTACCT
  CCTAGCAATGAACAAATTCT
• CAAGAAGATACTAGCACTATATGGACCAGTTTGTGTATCGTTACATTCGT
  CATTACAAAGTTTTGTAGCT
• TATCGAAGTGGTATTTATAATGATCCAAAATGTCCAACTAATGCAGAAAA
  AGTAAATCATGCGGTAATAG
• CGGTTGGTTATGGTGTCCAGAATGGGATGGAATATTTTATCATCAAAAAC
  AGTTGGGGCCCTACATGGGG
• TCAAAAAGGATACGGTCGTATTCGCGCTGGGGTATTTATGTGCGGTATTG
  GTCGTTTTTCGAACGTACCA
• ATCTTCAAATGA

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• gtgi214896764-1068 Haemonchus contortus
  cathepsin L cysteine protease (cpl-1) mRNA,
  complete cds
• ATGCTACGTCTGTTGTCGTTGGCGCTTCTATGCGCTGTAGTTTTAGCTAG
  TATTGATGGGTTCAGAAGGC
• ATGATCATGGCGTACGAGTGCACAGACAGAAAAGCCTTCGCCAAAAAATC
  GACGAGGCTTTCAATAAATG
• GGATGACTACAAGGAGACCTTTGGAAAGTCGTATGAACCGGATGAAGAGA
  ACGACTACATGGAGGCCTTT
• GTGAAGAACGTGATTCACATTGAGGAACACAATAAGGAACACCGTCTTGG
  TAGGAAAACATTTGAAATGG
• GTCTCAACGAAATTGCTGATTTGCCATTCTCACAATATCGAAAACTCAAC
  GGGTATCGTATGCGTCGTCA
• ATTTGGCGATTCCTTGCAGTCCAATGGTACCAAGTTTTTGGTTCCATTCA
  ATGTTCAGATCCCGGAATCT
• GTTGACTGGCGAGAGGAAGGACTTGTGACTCCAGTAAAGAATCAAGGAAT
  GTGTGGATCATGTTGGGCGT
• TCTCCTCTACTGGTGCTCTAGAAGGACAACATGCACGTGCCACTGGCAAG
  CTGGTATCCCTTTCCGAGCA
• AAATCTTGTCGATTGCTCAACGAAGTACGGAAACCATGGCTGCAACGGTG
  GTCTTATGGATTTGGCATTT
• GAATATATCAAGGAAAATCACGGTGTCGATACCGAAGATAGTTATCCATA
  CGTCGGAAGAGAAACGAAAT
• GTCATTTCAAGAGGAACGCTGTTGGAGCCGATGACAAGGGCTTTGTAGAT
  CTTCCTGAAGGCGATGAAGA
• GGCGTTGAAGAAAGCAGTTGCCACTCAAGGTCCAATTTCTATCGCTATTG
  ACGCTGGTCACAGGTCATTC
• CAGCTGTACAAGAAGGGAGTGTACTTTGACGAAGAGTGCTCGTCTGAAGA
  ACTGGATCACGGTGTTCTTC
• TCGTTGGATACGGTACTGATCCCGAGGCAGGAGATTATTGGCTTGTGAAG
  AACAGCTGGGGACCGACTTG
• GGGAGAGAAGGGATACATTCGCATTGCCCGTAACCGCAACAATCACTGCG
  GTGTTGCAACAAAGGCCAGC
• TACCCGCTCGTTTAG

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• gt(gi21483189lt1-53, 1121-1209, 1586-1725,
  1806-1910, 2049-2183, 2239-2385, 2448-2537,
  2616-2691, 2999-3107, 3188-3287) Dictyocaulus
  viviparus cathepsin L (cpl-1) gene, partial cds
• TTGCTTCTGTTCCTATGCGATTTAGCTTCAACTAAGATCGGTACACCTAG
  AAAGCACGGATTTTATTCGG
• AGAAACAGAAAAGTCTACGTCAGAGGATCGATGAGGCCTTTGGAAAATGG
  GATGAGTATAAGATCAAATA
• TGATAAACACTATGACCCTGAAGAAGAAAATGATTATATGGAGGCTTTTG
  TAAAAAACATGATCCACATC
• GAGGAGCATAATCATGAACACCGCTTGGGGCGAAAAACTTTCGAAATGGG
  ATTGAACAATATCGCTGATC
• TTCCTTTCTCGGAGTACCGCAAATTGAACGGTTACCGCCATCGTCGCTTA
  TTTGGTGACTCTATGCGTAA
• AAATGGCACGAAATTTTTAGTCCCTTTCAATGTCAAGGCGCCGGATTCAG
  TAGATTGGCGAGAACATAAT
• CTCGTTACTCCAGTAAAAAATCAAGGGATGTGTGGTTCCTGTTGGGCCTT
  CTCCGCAACTGGAGCTCTCG
• AAGGACAACATTTTCGTGCAACCGGAAAACTTGTTTCCTTGTCCGAACAG
  AATCTGGTGGATTGTTCTAC
• TAAATATGGAAACCATGGTTGTAACGGTGGTCTCATGGATTTGGCATTTG
  AATACATAAAAGATAATCAT
• GGTATAGATACTGAGGAGGGTTACCCTTATGTTGGCAAAGAGATGAGGTG
  CCACTTCAAAAAGAGGGACA
• TTGGAGCTGAAGATAGGGGGTTTGTAGATCTTCCAGAAGGAGACGAAGAT
  GCCTTGAAGGTCGCTGTCGC
• TACTCAGGGTCCTATTTCTATTGCCATTGATGCCGGTCATCGGTCTTTCC
  AGTTATATAAAAAAGGAGTT
• TATTTTGACGAGGAGTGTTCATCCGAAGAACTTGATCATGGAGTTCTTCT
  TGTGGGTTATGGCACTGATC
• CTGAAGCTGGCGACTACTGGATCATAAAGAATAGTTGGGGAACTAAATGG
  GGAGAAAAGGGTTACGTTCG
• TATCGCTCGGAATCGCAACAATCACTGCGGTGTGGCAACGAAGGCGAGTT
  ATCCGCTCGTCTAA

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Universal Primers

• Primers can be designed to amplify only one
  product.
• Primers can also be designed to amplify multiple
  products. We call such primers universal
  primers. For example, design primers to amplify
  all HPV genes.
• Strategy
• Align groups of sequences you want to amplify.
• Find the most conservative regions at 5 end and
  at 3 end.
• Design forward primer at the 5 conservative
  region.
• Design reverse primer at the 3 conservative
  regions.
• Matching forward and reverse primers to find the
  best pair.
• Ensure uniqueness in all template sequences.
• Ensure uniqueness in possible contaminant sources.

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Nucleic-acid Base Codes
Adapted from Mount, Bioinformatics Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY
(2001)
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• Catep1w 5'KTG YGG WKY VTG YTK KRY BTT CTC3'
• Catep2w 5'GTA WCC YTT YTS DCC CCA 3'
• Estos primers son las secuencias degeneradas de
  alinear los nematodes disponibles C.elegans,
  Brugian, Haemonchus, Dyctiocaulus

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Secuencia de DNA desconocida

• Extremos amino ó carboxilo terminal de la
  proteína codificada es conocida

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• P F T K
• CCn TTy ACn AAr
• Donde
• n A,C,G or Ty C,Tr A,G

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Our Focus the Helix-Coil Transition in DNA

• In particular, we focus on two related processes
• DNA melting
• B helix to two coils.
• DNA annealing
• two coils to a B helix.
• Understanding these
• aids in modeling more complicated transitions.
• e.g., many species.

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Stabilizing Interactions

• DNA B-Helix structure stabilized by
• hydrogen bonding between bases (minor).
• stacking between H-bonded
• base-pairs (primary).
• induced dipole moments in the p clouds of
  adjacent heterocyclic rings.
• stacking also sequesters hydrophobic rings.
• results in the characteristic helix shape.
• In DNA meltingthe helix destabilized
• generally implemented by increasing temperature,
  T.
• destabilizes the stacksunwinding the helix.
• unwound helix separates into free ssDNAs
  (coils).

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Monitoring the Helix-Coil Transition

• Degree of stacking experimentally observable
• Let QB mean fraction of stacked base pairs.
• Ultraviolet absorbance at 260 nm (A260)
• inversely proportional to QB.
• the hypochromicity.
• DNA melting accompanied by _at_ 40 increase A260.
• A260 vs. T yields QB vs. T (melting curve).

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DNA Melting Curves

• QB decreases monotonically from 1 to 0.
• sigmoidal shape indicates DNA melting is
  cooperative.
• Temp. at which QB ½ is the Melting temperature
  (Tm)
• Width (DT) is non-zero (e.g., for 10-mers, DT _at_
  10 oC).
• Melting curves of longer DNAs show more
  structure
• several independently melting regions (ATs less
  stable).
• melting curve then a combination of several
  sigmoids.

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DNA Renaturation

• Renaturation is the reverse of DNA melting.
• also called DNA annealing or hybridization.
• DNA renaturation is a much more complicated
  process

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DNA reassociation (renaturation)
Double-stranded DNA
Denatured, single-stranded DNA
Faster, zippering reaction to form
long molecules of double- stranded DNA
k2
Slower, rate-limiting, second-order process
of finding complementary sequences to
nucleate base-pairing
http//www3.kumc.edu/jcalvet/PowerPoint/bioc801b.p
pt
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Reversibility of DNA Melting

• Melting for short DNAs strictly reversible.
• Reversibility of DNA melting
• measured by a lack of hysteresis in the melting
  curve.
• DNA melting curve DNA renaturation curve.
• Validity of an equilibrium model of melting
  assumes
• melting slow enough to maintain equilibrium at
  each T.
• relatively slow heating/cooling
  (0.1-0.2oC/minute).
• failure to maintain equilibrium hysteresis in
  the melting curve.