Studies of Growth Hormone Gene
Sequence Studies
Byrne C. R, et al.1987 isolated
and sequenced the ovine growth hormone, together with about 1 kbp of DNA
flanking each end of the gene. The structure of the gene was similar to that
found for other growth hormone genes, particularly the bovine gene, and has a
primary transcript of 1792 bp, with five exons, and with intron sizes of 264
bp, 231 bp, 227 bp and 273 bp.
Orian et al. 1988 Sequenced
the sheep GH gene and the coding sequence predicted from that corresponded to
the cDNA sequence for pituitary GH, and found that this gene presumably codes
for the hormone expressed in the pituitary.
Guron C, et al. 1992 prepared
cDNA from the mRNA isolated from sheep anterior pituitary glands. On cloning
cDNA in E. coli, a clone coding full sequence of sheep pre-growth hormone was
determined. The sequence for the sheep growth hormone (GH) was in agreement
with the amino acid sequence of the protein determined previously except
positions 99 and 146.
Yamono Y, et al. 1988
isolated the cDNA that encodes goat growth hormone (gGH), from a goat pituitary
cDNA library. The cDNA was about 880 base pairs long and could code for a
polypeptide of 217 amino acids, The amino acid sequence homology between gGH
and the sequences of bovine GH, rat GH and human GH was 99, 83 and 66%,
respectively.
Woychik R. P., et al. 1982
Cloned and sequenced the bovine growth hormone gene. The sequence was about
1800 bp with four introns. The comparison of untanslated regions of the bovine,
human and rat growth hormone genes reveled many areas of highly conserved
sequence showing divergent evolution.
Wallis OC
& Wallis M. 1995 Cloned and characterized rabbit growth hormone encoding
gene. The amino acid sequence of rabbit GH was similar to that of pig GH and
other conserved mammalian GH, but differed markedly from the available sequences
of ruminant and primate GH. No evidence of cluster of GH like genes were found
as found in primates. This provides further support for the idea that, in
mammals, GH shows a slow underlying rate of evolution.
Duplication
of GH Gene in Caprine Ruminants (Sheep and Goat)
Valinsky et
al. 1990 concluded on the basis of studies on a restriction fragment length
polymorphism (RFLP), that there were two alleles at the GH
gene locus in sheep and goats. In sheep, in one allele (Gh1), the GH gene was represented
by a single copy (GH1 gene), while in the other (Gh2) the GH gene was
duplicated (GH2-N (5¢) and GH2-Z (3¢) genes). Restriction maps of the sheep Gh1 and Gh2 loci indicated
that the GH1, GH2-N and GH2-Z genes were all very similar. The sequences flanking the 5¢ ends of the GH1 and GH2-N genes were similar, but differ from
that flanking the GH2-Z gene. Individual animals were homozygous for Gh1 (i.e.
possessed 2 GH-like genes) or Gh2 (4 GH-like genes) or heterozygous, with one
copy of Gh1 and one of Gh2 (3 GH-like genes). The frequency of the Gh2 allele
was greater than that of the Gh1 allele.
Gootwine E. et al. 1993 found
PvuII restriction fragment length polymorphism at the growth hormone locus in
sheep carrying the GH2 allele where the gene was duplicated. By restriction
analysis and using the polymerase chain reaction they demonstrated that this
RFLP was due to a mutation at the PvuII site located in the second intron of
the 3' copy of the GH2 allele.
Gootwine et al. 1996
concluded that, in an animal homozygous for the Gh2 allele, only the GH2-N gene
was expressed in the sheep pituitary.
Ofir et al. 1997 Sequenced
each of the GH1, GH2-N and GH2-Z copies of the ovine GH genes of Awassi (a fat
tailed breed) and Romney breeds of sheep and compared them with DNA sequences
of pituitary and placental ovine GH expressed. (Gene Bank Accession numbers
AF002110-AF2129) The results showed that the structure of the single GH gene
copy of the ovine GH1 allele was highly conserved and the structure of the GH2-N
copy of the GH2 allele was slightly divergent from the GH1 copy and was
polymorphic, and that the GH2-Z gene copies were also polymorphic and
accumulated more substitutions both in the coding and the non-coding regions
then the GH2-N gene copy.
Lacroix et
al. 1996 studied growth hormones expressed in ovine placenta and found two GH
proteins, 22 and 28 kD. Sequences for three GH
related cDNAs derived from sheep placenta were also worked out. One of thoes,
coding for a protein identical to pituitary preGH, corresponded to the product
of the GH1 or GH2-N gene. The other two were very similar and were coding for a
protein differing from pituitary preGH at four amino acid residues, which
appeared to correspond to the product of the GH2-Z gene.
Yamano et al. 1991 isolated
two clones containing goat growth hormone (gGH) genes from goat genomic library
using goat growth hormone cDNA as a probe. One clone CgGH contained gGH1 gene,
and another clone, EgGH, contained gGH2 and gGH3 genes in tandem. DNA fragments
containing gGH1 gene, and that containing gGH2 and gGH3 genes, were estimated
to be allelic on the goat chromosome.
Seeburg PH. 1982 sequenced three
non-allelic genomic DNA fragments with each containing one member of growth
hormone gene. These genes were coding for the known polypeptide hormones,
growth hormone (hGH), chorionic somatomammotropin (hCS), and a yet unknown
protein, which differed from hGH in 13 positions. Each gene was structured into
five exons, the four introns occurring at identical positions, reflecting
recent gene divergence.
Barsh GS et al. 1983
determined the structure of the human growth hormone gene cluster over a
78-kilo base region of DNA by the study of two overlapping cosmids. There were
two growth hormone genes interspersed with three chorionic somatomammotropin
genes, all in the same transcriptional orientation. Analysis of the sequences
of the genes and identification of at least three different classes of
duplication units interspersed throughout the five gene cluster suggests that
the cluster evolved quite recently and that the mechanism of gene duplication
involved homologous but unequal exchange between middle repetitive elements of
the Alu family.
Chakravarti A. et al. 1984
detected six RFLPs in human growth hormone / human somatomammotropin gene
cluster, patterns of polymorphism and linkage disequilibrium suggested
independent origins of the human growth hormone gene cluster.
Hirt H. et al. 1987 obtained genomic
clones containing the closely related genes for human growth hormone (hGH) and
chorionic somatomammotropin (hCS), from genomic bacteriophage lambda and cosmid
libraries. The hGH/hCS locus contained two GH genes and three CS genes spanning
48 kb of DNA. Data obtained from that experiment combined with the nucleotide
sequences of all five GH and CS genes, indicated that the hGH/hCS gene locus
had evolved by duplication mechanism.
Chen et al. 1989 sequenced
the human chromosomal growth hormone locus contained on cloned DNA and spanning
approximately 66,500 bp, and it was found that this locus evolved by a series
of duplications and was found to contain five genes, which displayed 95%
sequence identity. The expression of each gene was examined by screening
pituitary and placental cDNA libraries by using gene-specific oligonucleotides.
According to this analysis, the hGH-N gene was transcribed exclusively in the
pituitary, whereas the other four genes (hCS-L, hCS-A, hGH-V, hCS-B) were
expressed only in placental tissue.
Bauman G. 1999 identified several
isoforms of growth hormone (GH) in humans. There were many reasons for this
heterogeneity. At the genetic level, two genes encode GH: GH-N, expressed in
the pituitary, and GH-V, expressed in the placenta. At the mRNA level, GH-N
undergo alternative splicing into 20K and 22K isoforms. Post-translationally,
22K GH undergo modifications, such as acetylation at its amino terminus,
deamidation, and oligomerization. The picture was complicated further in the
circulation, where GH binds to two GH-binding proteins, each with different
affinities for the GH isoforms.
Golos et
al.1993 screened Rhesus monkey pituitary and placental cDNA libraries
for hCS-hybridizing clones and found that there were at
least five genes that code for GH-like proteins, one expressed in the pituitary
and four in the placenta, although these may not be exactly equivalent to the
human GH-like genes.
Ber et al.
1993 reported the sequences of two growth hormone encoding genes from tilapia
fish (Tilapia nilotica). Sequencing data indicated that it was a consequence of
a relatively recent duplication event. The two genes were highly homologous,
having a similar intron (five)/exon (six) arrangement, and both encode an
identical polypeptide.
Physiological Effects of GH Copy
Number
Fleming et
al.1997 investigated growth hormone (GH) gene expression in pituitaries of
lambs from flocks selected for high (fat) or low (lean) back fat depth, which
were also homozygous for a single GH gene allele, heterozygous or homozygous
for duplication in the GH gene. It was conclude
that the pituitary glands of lean sheep were bigger and have an increased GH
content, but appeared to contain similar concentrations of GH mRNA and
immunoreactive GH as the pituitaries of fat sheep. The presence of the GH gene
duplication in sheep had little measurable effect on the expression and storage
of GH in the pituitary.
Gootwine et
al. 1997 studied the effect of growth hormone gene copy number (two three
or four for different genotypes of GH gene) on growth parameters, plasma GH
profile and response to GHRH. No significant effect of GH genotype on any
growth parameter or body composition was found. But Significant differences
were found between GH genotype and response to GHRH, this suggested that
polymorphism in GH gene copy number may have its physiological implications for
the functioning of the GH axis.
Balatskii VN
and Pocherniaev KF. 1995 investigated Restriction fragment length polymorphism
of growth hormone gene in Big White and Mirgorod pigs. A polymorphous BsuRI
restriction site was found within an amplified gene-region near 5-end. It was
localized within the first intron, 1.6 b.p. higher than exon 2 was. Homozygous
as well as heterozygous animals according to variants of the gene were found.
Fu J. et
al. 1995 studied MspI polymorphism in THY1 gene, encoding a cell surface
glycoprotein predominantly expressed in brain and peripheral nerves. Two
primers derived from the sequences flanking the polymorphic MspI site in intron
2 of the human THY1 gene were selected for RCP to amplify a 566 bp fragment
that spans the MspI polymorphism. Polymorphism was detected by MspI digestion
of the PCR product.
Polymerase Chain Reaction
Rychlik W.
(1995 a) explained the importance of careful selection of primers as most
important factor affecting the quality of PCR.
In general, the longer the PCR product the more difficult it was to
select efficient primers and set appropriate designing primers, and in general,
the more DNA sequence information was available, the better the chance of
finding an optimal primer pair. Efficient primers could be designed by avoiding
the following flaws: primer-dimer formation, self-complementarity, too low Tm
of the primers, and/or their incorrect internal stability profile.
Nei M and Li
WH. 1979 developed a mathematical model for the evolutionary change of
restriction sites in mitochondrial DNA. Formulas based on this model were
presented for estimating the number of nucleotide substitutions between two
populations or species. To express the degree of polymorphism in a population
at the nucleotide level, a measure called "nucleotide diversity" was
proposed.
Bains W.1994
examined the constraints of the design of oligonucleotide probes that were
meant to hybridize to different unique sites in human genomic DNA. In 522 kb of
human genomic DNA, 75% of 12-base and 89% of 22-base were unique, as opposed to
90% and 100% as expected of unstructured DNA, and this was not due solely to
repetitive elements in the DNA. Hybridization in TMAC to reduce A+T content
effects on melting temperature allows only 90% of unique targets to be
hybridized under one set of conditions if a 2 degrees C difference between
matched and mismatched sequences was required. This suggested that probe;
hybridization applications would be harder than previously suggested.
Kwok S. 1990
investigated the effects of various primer-template mismatches on DNA
amplification of an HIV-1 gag region by PCR. Single internal mismatches had no
significant effect on PCR product yield while those at the 3'-terminal base had
varied effects. A:G, G:A and C:C mismatches reduced overall PCR product yield
about 100-fold, A:A mismatches about 20-fold. All other 3'-terminal mismatches
were efficiently amplified. It was noted that mismatches of T with either G, C,
or T had a minimal effect on PCR product yield. Double mismatches reduced PCR
product yield dramatically, however presence of a mismatched T at the
3'-terminus, allowed significant amplification even when coupled with an
adjacent mismatch. Furthermore, even two mismatched Ts at the 3'-terminus
allowed efficient amplification.
Thompson JD. et al. 1994 improved the
sensitivity of the commonly used progressive multiple sequence alignment
method for the alignment of divergent protein sequences. Firstly,
individual weights were assigned to each sequence in a partial
alignment. Secondly, amino acid substitution matrices were varied at
different alignment stages according to the divergence of the
sequences to be aligned. Thirdly, residue-specific gap penalties and
locally reduced gap penalties in hydrophilic regions encourage new
gaps in potential loop regions rather than regular secondary structure.
Fourthly, positions in early alignments where gaps have been opened
receive locally reduced gap penalties to encourage the opening up of new
gaps at these positions. These modifications were incorporated into a new
program, CLUSTAL W that was freely available.
Thompson JD. et al. 1997
developed CLUSTAL X, a new windows interface for the multiple sequence
alignment program CLUSTAL W. The new system was easy to use, providing an
integrated system for performing multiple sequence and profile alignments and
analysing the results. A versatile sequence colouring scheme allowed the user
to highlight conserved features in the alignment. Pull-down menus provided all
the options required for traditional multiple sequence and profile alignment.
CLUSTAL X worked well on many operating systems.
Altschul SF et
al. 1997 described the BLAST programs as widely used tools for searching
protein and DNA databases for sequence similarities. The authors described a
variety of statistical refinements, which decreased execution time of the BLAST
programs while enhancing their sensitivity to weak similarities. A new
criterion for triggering the extension of word hits, combined with a new
heuristic for generating gapped alignments, yields a gapped BLAST program that
runs at approximately three times the speed of the original. In addition, a
method was introduced for automatically combining statistically significant
alignments produced by BLAST into a position-specific score matrix, and
searching the database using that matrix.
Template DNA
Gustafson at el. 1993 showed
that lengthy denaturation times of template DNA ranging from 1 to 7 min at pH
7.0-8.0, that were often employed prior to the start of a PCR reaction,
resulted in marked degradation of the template. That can result in a
significant reduction in the yield of PCR products larger than 500 bp, by up to
99%. This effect was demonstrated for both complex genomic template DNA, and
also for a 2691-bp linear piece of template DNA using both a rapid hot-air
thermocycler and a conventional block thermocycler. This decrease in product
yield was likely due to the increased degradation of the template or target DNA
as a result of pre-amplification denaturation (PAD). Therefore (PAD) was not
recommend for amplifying larger pieces of DNA, irrespective of the starting pH
of the template solution.
Vazquez and
Steinberg 1999 showed that genomic DNA preparations derived from
mammalian cells can often exhibit poor template activity in PCR, particularly
when carried out on target sequences present at low copy number. Using genomic
DNA bearing SV40 sequences integrated into host chromosomal DNA at low copy
number as a target, they show that template efficiency can be dramatically
enhanced after treatment of the genomic template with restriction enzymes for
varying periods of time. Results indicated that optimal digestion time varied
for each enzyme.
Chien A et
al. 1976 purified a stable DNA polymerase (EC 2.7.7.7) with a temperature
optimum of 80ºC from the extreme thermophile, Thermus aquaticus. The
enzyme was free from phosphomonoesterase, phosphodiesterase and single-stranded
exonuclease activities. Maximal activity of the enzyme requireed all four dNTPs
and activated calf thymus DNA. An absolute requirement for divalent cation
cofactor was satisfied by Mg2+ or to a lesser extent by Mn2+.
The pH optimum was 8.0 in tris-HCl buffer. The molecular weight of the enzyme
was estimated approximately 63,000 to 68,000. The elevated temperature
requirement, small size, and lack of nuclease activity distinguished this
polymerase from the DNA polymerase of Escherichia coli.
Kaledin AS et
al. 1980 isolated a 62 kD DNA polymerase from the thermophylic bacteria Thermus
aquaticus YT-1. The enzyme does not contain contaminant exo and
endonuclease activities and had a temperature optimum on the DNA templates at
70 degrees and that on RNA matrices at 50 degrees. The maximal activity of the
enzyme required the presence of bivalent cations (Mg2+ or Mn2+)
0,1-0,2 M KCl or NaCl, all dNTPs and template in the incubation mixture. The
enzyme was active when "activated" DNA, poly (dA)-poly (dT), poly
(dA)-oligo (dT) 10 and poly (rA)-oligo (dT)10 were used as templates and was
inactive on the native and denatured DNAs as well as on the native molecules of
RNA and poly (rC)-oligo (dG) 12-180.
Saiki RK et
al. 1988 used a thermostable DNA polymerase in an in vitro DNA
amplification procedure, the polymerase chain reaction. The enzyme, isolated
from Thermus aquaticus, greatly simplified the procedure and, by
enabling the amplification reaction to be performed at higher temperatures,
significantly improved the specificity, yield, sensitivity, and length of
products that could be amplified. Single-copy genomic sequences were amplified
by a factor of more than 10 million with very high specificity, and DNA
segments up to 2000 base pairs were readily amplified. In addition, the method
was used to amplify and detect a target DNA molecule present only once in a
sample of 10(5) cells.
Rees W.A., et
al. 1993 showed that the amino acid analogue betaine shares with
small tetraalkylammonium ions the ability to reduce or even eliminate the base
pair composition dependence of DNA thermal melting transitions. The
"isostabilizing" concentration of betaine (at which AT and GC base
pairs were equally stable) was approximately 5.2 M. Betaine exerted its
isostabilizing effect without appreciably altering the conformation of
double-stranded DNA from the B form. The presence of > 5 M betaine also did
not greatly changed the behavior of DNA as a polyelectrolyte; this lack of
effect on electrostatic interactions was expected because betaine exists as a
zwitterion near neutral pH.
Henke W., et
al, 1997 showed that Betaine
improved the co-amplification of the two alternatively spliced variants of
the prostate-specific membrane antigen mRNA as well as the amplification of the
coding cDNA region of c-jun. It was suggested that betaine improved the amplification
of these genes by reducing the formation of secondary structure caused by
GC-rich regions and, therefore, may be generally applicable to improve the
amplification of GC-rich DNA sequences.
Waleed Abu Al-Soud and Peter Rådström in 2000
investigated the effects of 16 amplification facilitators to
enhance DNA amplification in the presence of blood, feces, or meat.
The addition of 0.6% (wt/vol) bovine serum albumin to reaction
mixtures containing Taq DNA polymerase reduced the inhibitory
effect of blood and allowed DNA amplification in the presence of 2%
instead of 0.2% (vol/vol) blood. Taq DNA polymerase was
able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol)
feces and 4% instead of 0.2% (vol/vol) meat, The single-stranded DNA
binding T4 gene 32 protein (gp32) had a relieving effect similar to
that of BSA, they also found that addition of 11.7% (wt/vol) betaine allowed
Taq DNA polymerase to amplify DNA in the presence of 2% (vol/vol)
blood, while the addition of proteinase inhibitors allowed DNA
amplification in the presence of 4% (vol/vol) feces. When various
combinations of betaine, BSA, gp32, and Proteinase inhibitors were
tested, no synergistic or additive effect was observed. The effects
of facilitators on real-time DNA synthesis instead of conventional
PCR were also studied.
Kreader C.A.1996 studied the benefits of adding bovine serum
albumin (BSA) or T4 gene 32 protein (gp32) to PCR, with reaction
mixtures containing substances that inhibit amplification. Whereas 10
to 1,000 fold more FeCl3, hemin, fulvic acids, humic acids, tannic
acids, or extracts from feces, freshwater, or marine water were
accommodated in PCR when either 400 ng of BSA per microl or 150 ng
of gp32 per microl was included in the reactions, neither BSA nor
gp32 relieved interference significantly when minimum inhibitory
levels of bile salts, bilirubin, EDTA, NaCl, sodium dodecyl sulfate,
or Triton X-100 were present. Use of BSA and gp32 together offered no
more relief of inhibition than either alone at its optimal level, and neither
protein had any noticeable effect on amplification in the absence of
inhibitors.
Paabo S. et al. 1988 amplified pieces of
mitochondrial DNA from a 7000 year old human by the polymerase chain reaction
and sequenced them. Albumin and high concentrations of polymerase
were required to overcome a factor in the brain extract that
inhibited amplification. For this and other sources of ancient DNA,
authors found an extreme inverse dependence of the amplification efficiency
on the length of the sequence to be amplified. This property of ancient
DNA distinguished it from modern DNA and thus provided a new criterion
of authenticity for use in research on ancient DNA.
Certain organic
solvents, such as DMSO and betaine, had been reported to enhance PCR
amplification, particularly for hard to amplify high GC templates. Chakrabarti
R. and Schutt CE. 2002 studied two groups of compounds amides and sulfones and
discovered several potent PCR enhancers, often outperforming DMSO. They
identified tetramethylene sulfoxide as the most potent sulfur-oxygen compound
in the enhancement of PCR amplification and as one of the most potent PCR
enhancers currently known.
Choi JS et al. 1999 described
the use of dimethyl sulfoxide (DMSO) for sequencing of high GC content
templates. It was found that 5% (v/v) of DMSO in the reaction mixture recovered
sequencing signal intensity with reduced frequency of ambiguous bases. DMSO has
been empirically introduced to enhance the efficiency of PCR in GC-rich
templates. However, the underlying mechanism of improved cycle sequencing by
DMSO was unknown. Most of methyl nitrogen ring containing chemicals did not
improve sequencing accuracy, whereas only glycerol mimicked the positive effect
of DMSO by the same extent.
Baskaran N. et al. 1996 developed a PCR method
for uniform amplification of a mixture of DNA templates differing in
GC content, using the two enzyme approach (Klentaq1 and Pfu DNA
polymerase) and a combination of DMSO and betaine. This method was
applied to amplify the CGG repeat region from the fragile X region.
Watanabe M. et
al. 1996 established a simple and reproducible assay of polymerase chain
reaction (PCR), to detect trinucleotide repeat expansion for Huntington's
disease (HD) using a new DNA polymerase and buffer system. The system consists
of an extremely heat stable DNA polymerase (Pfu), and a buffer supplemented
with ammonium sulfate and dimethyl sulfoxide. Previous methods to amplify
expanded alleles for HD have been very complex in PCR conditions, and of low
reproducibility because of repetitive sequences around the primer sequences.
With the present method, strong bands for the disease alleles were easily and
reproducibly visible.
Olive DM et
al. 1989 established a PCR assay for direct detection of human
cytomegalovirus (HCMV). The efficiency of the amplification reaction was
examined by using three different buffers and concentrations of dNTPs. The PCR
assay was most efficient with a reaction mixture containing 17 mM ammonium
sulfate, 67 mM Tris hydrochloride (pH 8.5), 7 mM MgCl2, 10 mM
2-mercaptoethanol, 170 micrograms of bovine serum albumin per ml, and each dNTP
at a final concentration of 1.5 mM. The viral DNA was successfully amplified
directly from 5 microliters of urine preheated at 93ºC for 30 min.
Demeke and
Adams (1992) surveyed of the inhibitory effects of various plant
polysaccharides on PCR amplification of a 974-bp section of rbcL in Spinach and
revealed that most of the polysaccharides tested were not inhibitory. In
contrast, two of the acidic polysaccharides (dextran sulfate and gum ghatti)
were inhibitory. The addition of 0.5% Tween 20 reversed the inhibitory effects
of gum ghatti (polysaccharide: DNA ratio of 500:1). The inhibitory effect of
dextran sulfate (50:1) could be reversed by the addition of Tween 20 (0.25 or
0.5%) DMSO (5%), or polyethylene glycol 400 (5%) but none of these three
additives were effective at a 100:1 ratio of dextran sulf ate/DNA.
Varadaraj K,
Skinner DM. 1994 describe conditions that improve the specificity of
amplification of a GC rich (57% G + C) DNA by PCR. Under standard conditions a
368-bp segment of the satellite DNA of the Bermuda land crab was not amplified
specifically. To establish optimal conditions two enzymes, AmpliTaq DNA
polymerase and AmpliTaq DNA polymerase, Stoffel fragment (SF) along with and a number of co-solvents were studied.
Reagents that improved specificity of the amplified product were formamide,
glycerol, DMSO, Tween-20 and NP-40; on the other hand, urea, ethanol and
1-methyl-2-pyrrolidone (NMP) inhibited amplification. Of the two enzymes, SF
was more specific and efficient.
Kovárová M. and Dráber P (2000) compared the
ability of several Tetramethylammonium (TMA) derivatives and some other
reagents to increase the specificity of PCR and to improve the yield
of amplification. A novel combination of the enhancer TMA and oxalate
as anion was demonstrated to be a powerful enhancer of PCR. Addition
of 2 mM TMA oxalate to the PCR mixture decreased the formation of
non-specific DNA fragments and increased the yield of specific PCR
products.
Breslauer KJ. et al. 1986
reported the complete thermodynamic library of all 10 Watson-Crick DNA
nearest-neighbor interactions. This thermodynamic data was used to calculate
temperature dependent behavior of any DNA duplex from its sequence. Authors
illustrated a method of calculation by using the nearest-neighbor data to
predict transition enthalpies and free energies for a series of DNA oligomers.
These predicted values were in excellent agreement with the corresponding
values determined experimentally. This agreement demonstrated that a DNA duplex
structure thermodynamically can be considered to be the sum of its
nearest-neighbor interactions.
Peuschel KE. 2000 worked with
megaprimers of length 450-770 bp, optimal annealing of the megaprimer to the
template-DNA was shown to occur at unexpectedly low temperatures under conditions
suboptimal for amplification of the elongated megaprimer product by the short
flanking primers, although very high annealing temperatures had been expected
to give best results. The finding contradicted the rule that annealing
temperatures should be just below the melting temperature of a given primer,
and leaded to the conclusion that the curves of melting and annealing
temperatures diverge rather than running in parallel.
Owczarzy R. et
al. 1997 compared the 11 published sets of nearest neighbor sequence
dependent thermodynamic parameters for DNA, and demonstrated use of the
nearest-neighbor sets in predicting tm from the DNA sequence. The authors also
assessed the ability of the nearest neighbor parameters to provide accurate
predictions of experimental tm's of short duplex DNA oligomers.
Rychlik W et
al. 1990 experimentally determined the optimal annealing temperature
(TaOPT) values for several primer template pairs and develop a method for its calculation.
At both sub and super optimal Ta values, non-specific products may be formed,
and the yield of products was reduced. Optimizing the Ta was especially
critical when long products were synthesized or when total genomic DNA was the
substrate for PCR. The TaOPT was found to be a function of the melting
temperatures of the less stable primer-template pair and of the product. The
fact that experimental and calculated TaOPT values agree to within 0.7 degree C
eliminateed the need for determining TaOPT experimentally.
Ou CY et
al. 1991 described UV irradiation as a simple and efficient way to minimize
contamination or false positivity in routine PCR tests. They characterized the
effect of UV irradiation on DNA template, primers, deoxynucleoside triphosphate
and Taq polymerase. Reduction of the HIV DNA templates in polypropylene
microcentrifuge tubes by more than 1000-fold can be achieved by UV irradiation.
The sensitivity of the primers was sequence and concentration dependent. Taq
polymerase was highly UV sensitive, whereas deoxynucleotide triphosphate was
relatively UV resistant.
Prince AM
and Andrus L. 1992 investigated the use of sodium hypochloride (Clorox), in
comparison to concentrated HCl, for PCR sterilization. Ten percent Clorox was
found to eliminate all ethidium bromide-stainable DNA and to prevent PCR
amplification of a 600-bp DNA segment within one minute of template treatment.
RNA was similarly destroyed. By contrast, even 2.0 N HCl did not destroy DNA
detectable by PCR within five minutes.
Linz U et
al. 1990 optimized PCR parameters using different templates, together with
6 sets of primers, the following optimal conditions were found: the DNA should
be linearized, primer concentration 0.1-0.2 mumol/L, magnesium ion
concentration <2 mmol/L. pH 8.5-9.0. 25 cycles were sufficient. For
fragments greater than 103 bases the elongation time should be 5
min. The elongation temperature was not found critical and could vary between
50 and 70ºC. The hybridization temperature was used to control the specificity
of the polymerase chain reaction and, finally, mismatches at the 3' end of the
primer could totally inhibit the amplification.
Rychlik W.
(1995 b) tested effects of the primer concentration, annealing temperature,
salt and solvent concentrations on PCR yield. Priming was detectable when the
3' terminal portion of the partially mismatched primer formed a continuous
duplex more stable than -11 kcal/mol with the target DNA. In the presence of
low magnesium ion concentrations, priming was significantly reduced; glycerol
(5%) and formamide (2.5%) had only a slight effect (Taq DNA polymerase).
Oligonucleotides that were GC rich at their 3' ends exhibited high priming
efficiency but were also prone to false priming, since the shorter fragments of
their 3' ends were stable enough to serve as primers.
Harris S and
Jones DB. 1997 assessed the parameters, which influence DNA amplification
efficiency and specificity. As no single protocol was appropriate for all
situations and optimizing PCR required evaluation of the denaturation,
annealing and extension temperatures, the number of cycles performed, and the
primer, magnesium chloride, dNTP, Taq DNA polymerase and DNA template
concentrations. The important parameters for efficient, specific amplification
were denaturation time and temperature, stringent annealing temperatures and
magnesium chloride concentration. The importance of DNA concentration was found
to depend upon the source from which the DNA was extracted.
Nasri M et
al. 1985 worked out the activity of Pvu II and found that the
substrate specificity of PvuII endonuclease was relaxed in the presence of
dimethyl sulfoxide. The new recognition sequences cleaved in pBR322 DNA have
been found to be CCGCTG, CATCTG, CAGATG, CAGGTG and CAGCGG.
Nasri M. and Thomas D. 1987 found the restriction endonuclease PvuII that cleaves the sequence
CAGCTG, decreased its substrate specificity in the presence of organic
solvents. Thirty three sites were identified on the nucleotide sequence of
pBR322 DNA. The new recognition sequences were shown to be AAGCTG,
GAGCTG, CNGCTG, CANCTG, CAGNTG, CAGCNG, CAGCTC and CAGCTT. PvuII
recognized and cleaved degenerate sequences, which differ from the
standard PvuII sequence CAGCTG at only one of the recognition site.
The optimum incubation medium for PvuII activity was found to be:
10-50 mM Tris-HCl, pH 8.5, 12-15 mM MgCl2, 50 mM NaCl, 10% ethanol +
10% dimethylsulfoxide.
George J and Chirikjian JG. 1982
studied the effect of glycerol on the specificity of DNA cleavage by the
restriction endonuclease BamHI. In addition to the canonical G decreases from
G-A-T-C-C site, BamHI cut DNA at several other sites, number of BamHI.1 sites
in simian virus 40 and pBR322 was determined to be 13 for each DNA. Those
included G decreases from G-A-A-C-C, G decreases from G-C-T-C-C, G decreases
from G-G-T-C-C, and G-A-A-T-C-C with the complementary strand sequence
assignments of G-G-T-T-C-C, G-G-A-G-C-C, G-G-A-C-C-C, and G-G-A-T-T-C. The
relaxation in specificity was related to hydrogen bond acceptor and donor sites
in the recognition sequence.
Nasri M. and Thomas D .1986 studied that under the standard
reaction conditions, the restriction endonuclease HindIII cleaves
double-stranded DNA, within the recognition sequence A decreases
AGCTT. In the presence of Dimethyl sulfoxide the substrate
specificity of this enzyme was reduced and cleavages occurred at
additional sites. They had determined the secondary sites in pBR322
DNA under relaxed conditions and found that it cleaveed: G decreases
AGCTT, A decreases GGCTT, A decreases TGCTT, A decreases ATCTT, A
decreases AGCCT, A decreases AGCAT, A decreases AGCGT, A decreases
AGCTC, producing fragments with cohesive ends.