HAEMOCHROMATOSIS DETECTION USING PCR-RFLP INTRODUCTION Hereditary Haemochromatosis (HH), first described in 1865, is a genetic disorder of metabolism, characterized by progressive iron overload resulting from abnormalities in intestinal iron absorption and or release of iron from cells. It is an recessive disorder, where the body accumulates excessive iron, which is deposited in a variety of organs. Iron cannot be excreted, thus, the excess builds to toxic levels in tissues of major organs such as the liver, heart, pituitary, thyroid, pancreas, lungs, and (joints).
These organs cease to function adequately and eventually become diseased. Serious illnesses such as diabetes, cirrhosis, hematoma, , cardiomyopathy and arthritis may be a consequence of this disease. It affects one in every three hundred Caucasians, and one in nine is a carrier, hence, making its early detection vital.
The gene responsible for HH (HLA-H) was recently identified on the short arm of chromosome 6 and is thought to be mainly caused by a mutation of a gene called HFE, which allows excess iron to be absorbed from the diet. This mutation is known as C 282 Y. A single point mutation occurs, in which the amino acid cysteine at position 282 changes to a tyrosine. To develop two genes, one from each parent, are required to be C 282 Y. However, not everyone with the mutation may develop the disease and it may occur if only one C 282 Y gene is present (4).
77. 5% of affected individuals have two copies of the C 282 Y mutation, one inherited from each parent, while about 4% have a single copy of the mutation and one normal HFE gene. First proposed in early 1970’s, Polymerase Chain Reaction (PCR) has been identified as a simple, robust, speedy, and most of all, flexible method that can be used to detect. In this technique, specific DNA sequences are amplified for the detection of mutations that may be present, allowing early diagnosis of hereditary (see figure 1).
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It is a major development in the analysis of DNA and RNA. The requirements of the reaction are simple, consisting of to provide both the energy and nucleosides for the synthesis of DNA, template, primer, DNA polymerase, and buffer containing magnesium.
The crux of the PCR procedure involves three steps, including denaturation at high temperatures, annealing of primers, and extension, which are repeated for 30-40 cycles. The aim of this experiment was to use a PCR-RFLP to determine the presence of HLA-H gene responsible for hereditary. The genomic DNA containing the Cys 282 Tyr mutation was amplified by PCR and the mutation was detected in several individuals. In addition, it is also known that this mutation results in the establishment of a new RsaI site into the DNA, which was identified by restriction digestion of the PCR product. Moreover, vital variables, such as magnesium ion concentration, primer annealing temperature and template concentration, were also examined to establish their relationship with the efficiency and specificity of target DNA by the PCR.
METHODS The procedures dictated in ‘Biochemistry Genes and Disease’, Practical manual. 2004 pages 21-27 were followed. The only amendment that was in experiment one. The step “Overlay reactions with 100 ul mineral oil” was excluded. RESULTS Figure 1 The PCR product obtained from the amplification of HLA-H Genomic DNA. The following could be seen from the electrophoresis analysis: .
Lane 1 depicts bands for p UC 19/HpaII markers… Lanes 2 & 3 display the heterozygous and homozygous bands (alleles) respectively… Lane 4-10 show bands for ‘Wild Type’, which represents two normal alleles, thus not being exposed to the HH disorder… Lanes 5-7 depict the effects of varying magnesium concentration, as tested in experiment 1. At 0 ul, there is no band present which can be seen in Lane 5.
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However, as magnesium is added, in Lanes 6 and 7, bands appear at varying intensity. At 1 ul an ambiguous, band can be seen, which is smudged and undefined in nature, as seen in Lane 6. In Lane 7, at 2 ul, a bright, defined band is visible… Lanes 8-10 depict the results obtained as a consequence of altering the template concentration, as dictated in experiment 2. At 1 ul in Lane 8 results illustrates faint, unclear band. However, its clarity is seen to improve and seen being optimal in Lane 9, at 3 ul of the template.
Additional DNA in Lane 10, at 10 ul, shows a compromise in the quality of the band. The band appears to be bright, yet inadequate in thickness. Figure 2 Electrophoresis analysis of Wild Type using annealing Temperature Dependence. This illustrates the results obtained for experiment 3, in which the effects of temperature on PCR were observed: . Lane 1 depicts bands for p UC 19/HpaII markers… Lane 2 illustrates the presence of unclear, clustered bands, lacking any definition, when treated at 45 C.
Figure 3 Gel electrophoresis analysis of RsaI digestion of HLA-H PCR product. This figure depicts electrophoresis of Heterozygous and Homozygous alleles compared to standard markers… Lane 1 depicts bands for p UC 19/HpaII markers… Lane 2 displays two bands at 260 bp and 135 bp, which represent the two normal alleles of the ‘Wild type’… Lane 3 illustrates the presence of 260 bp, 135 bp, 105 bp and 30 bp bands, which exemplifies the presence of one normal and one mutated allele, proving it to be heterozygous in nature… Lane 4 shows bands at 260 bp, 105 bp and 30 bp.
This depicts the presence of two mutated alleles, and fulfills the requirements for being homozygous in nature. Figure 4 The class PCR product obtained from the amplification of HLA-H Genomic DNA. Table 1 a & b. Final concentrations of each reagent in the PCR reaction. REAGENT dNTPs Tris buffer Primers CONCENTRATION 0. 44 mM 20.
02 mM 0. 001012 mutable 1 b REAGENT Mg 2+ (vol = 1. 0 ul) Mg 2+ (vol = 2 ul) DNA (1 ul) DNA (3 ul) DNA (10 ul) CONCENTRATION 2. 0 mM 4. 0 mM 0. 8 ng 2.
4 ng 8 notable 2 Comparison of base pairs to mobility for p UC 19/HpaII Mobility (bp) Marker size (mm) 7 33111 24213 19017 14722 11132 67 The distance migration along the side was measured using a ruler to assist in constructing a calibration curve to determine the sizes of the DNA fragments produced by PCR. See Figure 5 for the graph. A mobility V’s log Marker size was plotted to gain this. DISCUSSION Affecting 1 in 200 Australians of Anglo-Celtic descent, Hereditary Hemochromatosis (HH) is one of the most common genetic disorders with high morbidity and mortality which is preventable if diagnosed early. In 1996, Feder et al. drew a connection between mutations in a gene and hereditary.
PROBE-BASED DETECTION SYSTEMS14 Hybridization probes (also called FRET probes)16 MELTING CURVE ANALYSIS16 Multiplex real-time PCR18 APPLICATIONS OF REAL TIME PCR18 GENE EXPRESSION ANALYSIS18 SNP GENOTYPING19 HIV DETECTION19 CYSTIC FIBROSIS (CF) DETECTION:20 THE ADVANTAGES OF REAL-TIME PCR20 THE DISADVANTAGES21 REFRENCES21 REAL TIME PCR TRADITIONAL PCR The polymerase chain reaction (PCR) is one of ...
The HFE gene encodes a protein similar in structure to the MHC class 1 molecules. The protein encoded by the HFE gene interacts with the transferring receptor and is involved in the regulation of iron absorption. He found two point mutations which could repeatedly be found in patients with hereditary. The replacement of G (guanine) by A (adenine) at position 845 in the HFE gene causes in the protein a change from cysteine (C) to tyrosine (Y) at the amino acid position 282 (mutation C 282 Y).
This mutation occurs heterozygous ly in approx. 5% of the total population (6).
Over 90% of all patients with carry the allele C 282 Y homozygous ly. Following this characterization of the HFE gene in 1996, genetic testing for hereditary has become available. Studies indicate that susceptibility to hereditary have risen from 14 414 in 1999 to almost 30 000 in 2002.
Many symptoms are reversible, and normal lifespan is possible. Therapy after late detection is less effective, particularly if cirrhosis is present, and prognosis is guarded (11).
These implications and prevalence thus impose the need for the early detection and treatment for HH. Until the recent development of a DNA test, early diagnosis has been difficult. Despite the high prevalence of HH, most cases remain undiagnosed.
Testing has been based upon measurements of serum iron, iron binding capacity, transferring saturation, and ferritin concentration. However, these tests are imperfect, without clear ‘cut-offs’ for results indicating affected status. Liver biopsy has traditionally been used for definitive diagnosis, but this invasive procedure is performed late in the course of the disorder. A simple, non-invasive and inexpensive DNA test known as PCR (Polymerase Chain Reaction) analyses of targeted gene regions has been identified as a diagnostic method for the detection of HH (11).
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As mentioned, this technique is a primer extension reaction for amplifying specific nucleic acids in vito. It allow a short stretch of DNA (usually fewer than 3000 bp) to be amplified to about a million fold so that one can determine its size, nucleotide sequence, etc.
It is comprised of three major components, including Denaturation (94^0 C), Annealing (54^0 C), and extension (72^0 C), which are repeated for 30 or 40 cycles. It allows the amplification of genomic DNA containing the Cys 282 Tyr mutation and the identification of the mutation. Restriction digestion of the PCR product allows the detection of the new RsaI site on the DNA, which is a establishment of HH disease. The specificity and efficiency of the amplification of target DNA revolves around certain parameters that affect PCR (7), including components such as presence of magnesium, primer annealing temperature, and template concentration. ‘All thermos table DNA polymerases require free divalent cations’ (7) usually Mg 2+ for activity. dntps- Mg 2+ complexes interact with the sugar-phosphate backbone of nucleic acids and influence the activity of Taq DNA polymerase.
Hence, altering the concentration of Mg Cl 2 can lead to one Primer or template pair behaving significantly differently from another under Identical conditions (18).
A study corroborated this phenomenon, by suggesting that ‘Mg++ is known to play a critical role in amplification as it can affect DNA strand denaturation, primer annealing specificity and enzyme fidelity ‘. dNTPs and oligonucleotides bind Mg 2+, thus the molar concentration of the cation must exceed the molar concentration of phosphate groups contributed by dNTPs and primers. An imprecise magnesium concentration will reduces and in some instance, hinders the amplification of PCR product entirely. This was challenged in the experiment, in which the PCR reactions were observed in light of varying magnesium concentration.
Electropherosis analysis in Figure 1 endorsed the requirement of having an adequate magnesium concentration to obtain optimal PCR results. It depicts the varying samples of PCR that were electrophoreses on 4% high resolution arose gels for three varying Mg 2+ concentrations. At 0 ul in lane 5, no band is visible, thus signifying a lack of PCR product being formed. At 1 ul, in lane 6, a band can be seen, however, is ambiguous, smudged, distorted and undefined in nature.
Josh Hyman Per. 5 Plasmid Fusion & PCR The AMGEN Lab that we have been doing for the past two weeks consisted of two parts; Plasmid Fusion and PCR. Each one is a complicated procedure of genetic engineering, with our own cheek cells and E. Coli supplied by AMGEN. I will start by explaining the Plasmid Fusion lab. The Plasmid Fusion lab consisted of four major parts; plasmid digestion, gel ...
This exemplifies scarcity in the Mg 2+ concentration, which hinders the adequate formation of the PCR product. At 2 ul, in lane 7, a bright defined band is present, indicating that this concentration is optimal for gaining the PCR product. It could thus be concluded, that as the concentration of Mg 2+ was increased, more product was obtainable. However, its absence led to an absence of the product, hence making its presence vital for the PCR reaction to work.
Another variable that was challenged in this experiment was the primer annealing temperature. It has been found that the success of a PCR depends immensely on the specificity with which a primer anneals only to its target, and not non-target sequence, thus, making it imperative to optimize this molecular interaction. Whether a primer can anneal only to its complement or also to sequences that have one or more mismatches to the primer depends critically upon the annealing temperature. Generally, it follows a proportional relationship, as the higher the annealing temperature the more specific annealing of the primer to its perfect matched template and thus, the greater the likelihood of only target sequence amplification. On the contrary, the lower the temperature, the higher the presence of mismatches between template and primer, resulting in increased amplification of non-target sequences (18).
Another source corroborated this, however further stated that ‘if the annealing temperature is too high, the oligonucleotide primers anneal poorly, if at all, to the template, and the yield of amplified DNA is very low’ (7).
On the contrary, employing a temperature, which is below a critical level will result in ‘nonspecific annealing of primers’, causing ‘an amplification of unwanted segments of DNA’ (7), thus making the use of appropriate temperature for the annealing step critical and imperative. This phenomenon was challenged, in this experiment. As illustrated in Figure 2 when 45^0 C was used as the annealing temperature, clusters of smears were present, which lacked clarity or definition. This exemplifies the amplification of undesired segments of DNA, resulting in the lack of specificity, thus proving this annealing temperature inadequate for optimal results. However, class results in Figure 4, Lanes 6 and 7 depict the effects of using 62^0 C as the annealing temperature. Theoretically, Lane 6 should lack the presence of any band appearing, as the tube that was tested in this Lane, lacked any template DNA.
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This was observed in the gel electrophoreses in Figure 4, as only the tube with the DNA (Lane 7), gave a bright, defined band. Lanes 8 and 9 show the treatment under 45^0 C, with Lane 8 lacking DNA, thus showing no band. A band was visible in Lane 9, however, its size was inadequate, as it was too thick, again, supporting the findings in Figure 2, that 45^0 C was not an adequate temperature for optimal results. Thus it can be verified that the usage of 62^0 C gave optimal results, with specific annealing of the primer to its matched template. The amount of DNA template strongly influences the outcome of the PCR reaction. This association was evident in the results, in Figure 1, in Lanes 8-10, which showed the effect of varying the template concentration on the reaction.
At 1 ul in Lane 8, a faint, distorted band was visible. It lacked clarity and form. However, as the template concentration was increased to 3 ul in Lane 9, a clear, defined band was seen to illuminate, suggesting the optimal concentration for the template. When the concentration was increased further to 10 ul in Lane 10, there appeared to be a compromise in the quality of the band. The band appeared to be bright, yet inadequate in thickness.
Hence, these findings showed that 3 ul was the optimal template concentration to achieve adequate results. Any concentration lower than this compromised the clarity and visibility of the product, whilst increasing the concentration above this level, contrarily resulted in emergence of smudging and inadequate thickness of the product. As discussed, is an recessive disease caused by mutations in the HFE gene. To develop an inheritance of two defective gene (homozygous) from each parent is required to be C 282 Y. However, a person who inherits a defective gene from only one parent is heterozygous, and is a carrier but does not develop the disease. It has been discovered that this mutation results in the establishment of a new RsaI site into the DNA.
In the experiment, this was identified by restriction digestion of the PCR product. The genomic region which was amplified was 400 base pairs. In unaffected individual (Wild Type), a single RsaI site exists, which gives 260 base pairs and 135 base pairs bands when its cut. In an affected individual, an additional RsaI site eventuating in 260 base pairs, 105 base pairs and 30 base pairs bands (9).
In accordance to these findings, in Figure 3 it can be determined that Lanes 3 and 4, denoting heterozygous and homozygous respectively contained the Cys 282 Tyr substitution.
In Lane 3, the DNA is cut at 260 bp, 105 bp and 30 bp. However, since this individual has only one abnormal allele, he will not show the disease, but will be a carrier fro HH. In Lane 4, there is an addition of an RsaI site, showing a cut at 260 bp, 105 bp and 30 bp. This individual is seen to possess two abnormal alleles, suggesting, that he is homozygous, thus developing HH disease. REFERENCES web web web prevalent inherited%20 disease. htm 4 web web web Saybrook, Russell (2001): “Molecular cloning-a laboratory manual” (3 rd Ed) 8 M.
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