
Biochemistry
9th Edition
ISBN: 9781319114671
Author: Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher: W. H. Freeman
expand_more
expand_more
format_list_bulleted
Question
help me with part b part i and iii please . for iii. the hint is that A is constant so H and S cannot be in the same (). But I don't quite understand how to do it.
![(b) Shown below is a website from a company that sells oligonucleotides as
primers. They provide a discussion of how they calculate Tm for the oligos that you
might purchase using a table of AH and AS values that you add up for your
sequence. Describe how an equation like Eq. 1 can be derived from AG(Tm) = AH-
TAS and indicate:
(i) how your answer(s) to part (a) are incorporated into Eq. 1.
(ii) why is 1000AH used but not 1000AS ?
(iii) if there is any error in the formula shown
sigmaaldrich.com
Melting Temperature
☐ etermining the melting temperature, Tm, of an
oligo is essential for many applications such as
PCR, capture assays, mutagenesis, hybridization, and
sequencing. The exact Tm of your DNA can be deter-
mined only by empirical means.' However, the three
theoretical methods described here can approximate
the Tm. Selecting the appropriate equation depends
on your application.
What is Tm?
The Tm, or melting temperature, of an oligo is the temperature at
which 50% of the oligonucleotide and its perfect complement are
in duplex. Typically, annealing or hybridizations are performed at
5-10 °C below the Tm of a duplex. Failing to calculate the correct
Tm for an oligo could result in inappropriate duplex formation.
Primer mismatch, false priming, and background signal problems
could result if annealings and hybridizations are performed at
temperatures significantly below the oligo Tm. Using temperatures
well above the Tm of an oligo could result in reduced priming, or
no priming or hybridization. The main factors affecting Tm are salt
concentration, DNA concentration, the presence of denaturants
(formamide or DMSO), DNA sequence, and length.
Nearest Neighbor Method
At Sigma-Genosys, we use the nearest neighbor method to deter-
mine the Tm of oligonucleotides. This equation is considered to be
one of the more accurate derivations of Tm. The nearest-neighbor
method takes into account the actual sequence of your oligo,
whereas the other equations rely on the base composition to
calculate Tm. With the nearest-neighbor method, several oligos
with the same length and base composition, but differing
sequences, would have a different Tm. In Table 1, each of the
sequences contain 5 As, 5 Cs, 5 Gs, and 5 Ts. Notice the Tm varies
with each sequence despite the base composition being the same.
Table 1. Varying values of sequences with the same GC content.
The nearest-neighbor method incorporates certain variables such
as salt concentration and DNA concentration. Sigma-Genosys uses
conditions typically found in PCR applications (50 mM monovalent
salt and 0.5 μM primer). The nearest-neighbor equation for DNA
and RNA-based oligos is:
(1) Tm (1000AH/A+AS+ Rin (C/4)) - 273.15 + 16.6 log[Na+]
AH (Kcal/mol) is the sum of the nearest-neighbor enthalpy changes
for hybrids. A is a small, but important constant containing correc-
tions for helix initiation. AS is the sum of the nearest-neighbor
entropy changes. R is the Gas Constant (1.99 cal K-'mol), and C is
the concentration of the oligo. The AH and AS values for nearest-
neighbor interactions of DNA and RNA are shown in Table 2. In
many cases this equation gives values that are no more than 5 °C
from the empirical value. Please note that this equation includes a
factor to adjust for salt concentration.
Table 2. Thermodynamic parameters for nearest-neighbor
melting temperature formula.
DNA
RNA
Interaction
ΔΗ
AS
ΔΗ
AA/TT
-9.1
-24.0
-6.6
AS
-18.4
AT/TA
-8.6
-23.9
-5.7
-15.5
TA/AT
-6.0
-16.9
-8.1
-22.6
CA/GT
-5.8
-12.9
-10.5
-27.8
GT/CA
-6.5
-17.3
-10.2
-26.2
CT/GA
-7.8
-20.8
-7.6
-19.2
GA/CT
-5.6
-13.5
-13.3
-35.5
CG/GC
-11.9
-27.8
-8.0
-19.4
GC/CG
-11.1
-26.7
-14.2
-34.9
GG/CC
-11.0
-26.6
-12.2
-29.7
Initiation
0.0
-10.8
0.0
-10.8
Sequence (5'-3')
AAAAACCCCCGGGGGTTTTT
Tm
69.7
MW
LEN
GC
6103
20
50%
ACGTACGTACGTACGTACGT
57.2
6103
20
50%
GATCGATCGATCGATCGATC
64.5
6103
20
50%
ATATATATATCGCGCGCGCG
66.4
6103
20
50%](https://content.bartleby.com/qna-images/question/afade1a9-1f29-4d84-b55a-a57e5a303c8b/2695c27b-8319-4247-b06c-33c9cb3d3ca8/wvmqssq_thumbnail.jpeg)
Transcribed Image Text:(b) Shown below is a website from a company that sells oligonucleotides as
primers. They provide a discussion of how they calculate Tm for the oligos that you
might purchase using a table of AH and AS values that you add up for your
sequence. Describe how an equation like Eq. 1 can be derived from AG(Tm) = AH-
TAS and indicate:
(i) how your answer(s) to part (a) are incorporated into Eq. 1.
(ii) why is 1000AH used but not 1000AS ?
(iii) if there is any error in the formula shown
sigmaaldrich.com
Melting Temperature
☐ etermining the melting temperature, Tm, of an
oligo is essential for many applications such as
PCR, capture assays, mutagenesis, hybridization, and
sequencing. The exact Tm of your DNA can be deter-
mined only by empirical means.' However, the three
theoretical methods described here can approximate
the Tm. Selecting the appropriate equation depends
on your application.
What is Tm?
The Tm, or melting temperature, of an oligo is the temperature at
which 50% of the oligonucleotide and its perfect complement are
in duplex. Typically, annealing or hybridizations are performed at
5-10 °C below the Tm of a duplex. Failing to calculate the correct
Tm for an oligo could result in inappropriate duplex formation.
Primer mismatch, false priming, and background signal problems
could result if annealings and hybridizations are performed at
temperatures significantly below the oligo Tm. Using temperatures
well above the Tm of an oligo could result in reduced priming, or
no priming or hybridization. The main factors affecting Tm are salt
concentration, DNA concentration, the presence of denaturants
(formamide or DMSO), DNA sequence, and length.
Nearest Neighbor Method
At Sigma-Genosys, we use the nearest neighbor method to deter-
mine the Tm of oligonucleotides. This equation is considered to be
one of the more accurate derivations of Tm. The nearest-neighbor
method takes into account the actual sequence of your oligo,
whereas the other equations rely on the base composition to
calculate Tm. With the nearest-neighbor method, several oligos
with the same length and base composition, but differing
sequences, would have a different Tm. In Table 1, each of the
sequences contain 5 As, 5 Cs, 5 Gs, and 5 Ts. Notice the Tm varies
with each sequence despite the base composition being the same.
Table 1. Varying values of sequences with the same GC content.
The nearest-neighbor method incorporates certain variables such
as salt concentration and DNA concentration. Sigma-Genosys uses
conditions typically found in PCR applications (50 mM monovalent
salt and 0.5 μM primer). The nearest-neighbor equation for DNA
and RNA-based oligos is:
(1) Tm (1000AH/A+AS+ Rin (C/4)) - 273.15 + 16.6 log[Na+]
AH (Kcal/mol) is the sum of the nearest-neighbor enthalpy changes
for hybrids. A is a small, but important constant containing correc-
tions for helix initiation. AS is the sum of the nearest-neighbor
entropy changes. R is the Gas Constant (1.99 cal K-'mol), and C is
the concentration of the oligo. The AH and AS values for nearest-
neighbor interactions of DNA and RNA are shown in Table 2. In
many cases this equation gives values that are no more than 5 °C
from the empirical value. Please note that this equation includes a
factor to adjust for salt concentration.
Table 2. Thermodynamic parameters for nearest-neighbor
melting temperature formula.
DNA
RNA
Interaction
ΔΗ
AS
ΔΗ
AA/TT
-9.1
-24.0
-6.6
AS
-18.4
AT/TA
-8.6
-23.9
-5.7
-15.5
TA/AT
-6.0
-16.9
-8.1
-22.6
CA/GT
-5.8
-12.9
-10.5
-27.8
GT/CA
-6.5
-17.3
-10.2
-26.2
CT/GA
-7.8
-20.8
-7.6
-19.2
GA/CT
-5.6
-13.5
-13.3
-35.5
CG/GC
-11.9
-27.8
-8.0
-19.4
GC/CG
-11.1
-26.7
-14.2
-34.9
GG/CC
-11.0
-26.6
-12.2
-29.7
Initiation
0.0
-10.8
0.0
-10.8
Sequence (5'-3')
AAAAACCCCCGGGGGTTTTT
Tm
69.7
MW
LEN
GC
6103
20
50%
ACGTACGTACGTACGTACGT
57.2
6103
20
50%
GATCGATCGATCGATCGATC
64.5
6103
20
50%
ATATATATATCGCGCGCGCG
66.4
6103
20
50%
Expert Solution

This question has been solved!
Explore an expertly crafted, step-by-step solution for a thorough understanding of key concepts.
Step by stepSolved in 2 steps

Knowledge Booster
Similar questions
- what would be concerning about Oxycodone and acetaminophen combination, why interested in pursuing this topic.arrow_forwardMy question is Explain why you feel numbness when using a topically applied pain cream that contains lidocaine.arrow_forwardPlease don't provide handwritten solution .... In sickle cell anemia, only one substitution of glutamic acid by valine (one amino acid substitution), is known to be the most possible cause that changes normal red blood cells into sickle cell red blood cells. True Falsearrow_forward
arrow_back_ios
arrow_forward_ios
Recommended textbooks for you
- BiochemistryBiochemistryISBN:9781319114671Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.Publisher:W. H. FreemanLehninger Principles of BiochemistryBiochemistryISBN:9781464126116Author:David L. Nelson, Michael M. CoxPublisher:W. H. FreemanFundamentals of Biochemistry: Life at the Molecul...BiochemistryISBN:9781118918401Author:Donald Voet, Judith G. Voet, Charlotte W. PrattPublisher:WILEY
- BiochemistryBiochemistryISBN:9781305961135Author:Mary K. Campbell, Shawn O. Farrell, Owen M. McDougalPublisher:Cengage LearningBiochemistryBiochemistryISBN:9781305577206Author:Reginald H. Garrett, Charles M. GrishamPublisher:Cengage LearningFundamentals of General, Organic, and Biological ...BiochemistryISBN:9780134015187Author:John E. McMurry, David S. Ballantine, Carl A. Hoeger, Virginia E. PetersonPublisher:PEARSON

Biochemistry
Biochemistry
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:W. H. Freeman

Lehninger Principles of Biochemistry
Biochemistry
ISBN:9781464126116
Author:David L. Nelson, Michael M. Cox
Publisher:W. H. Freeman

Fundamentals of Biochemistry: Life at the Molecul...
Biochemistry
ISBN:9781118918401
Author:Donald Voet, Judith G. Voet, Charlotte W. Pratt
Publisher:WILEY

Biochemistry
Biochemistry
ISBN:9781305961135
Author:Mary K. Campbell, Shawn O. Farrell, Owen M. McDougal
Publisher:Cengage Learning

Biochemistry
Biochemistry
ISBN:9781305577206
Author:Reginald H. Garrett, Charles M. Grisham
Publisher:Cengage Learning

Fundamentals of General, Organic, and Biological ...
Biochemistry
ISBN:9780134015187
Author:John E. McMurry, David S. Ballantine, Carl A. Hoeger, Virginia E. Peterson
Publisher:PEARSON