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In this study, we investigate two families spanning three generations, wherein the genetic trait has been observed. However, due to sample mix-ups during storage, our primary objective is to accurately identify which samples belong to each family. To achieve this, we employ Amplified Restriction Fragment Length Polymorphisms (RFLPs), a molecular biology technique. RFLPs result in the gene splitting into four possible allelic forms during electrophoresis. Our findings confirm that the disease under investigation is autosomal recessive and not sex-linked. Nevertheless, we acknowledge the need for further testing to refine our understanding of this genetic trait.
A Restriction Fragment Length Polymorphism (RFLP) is a widely used technique in molecular biology that relies on the action of the enzyme 'restriction endonuclease.' This enzyme is capable of cleaving DNA at specific sites known as restriction sites, which are determined by particular nucleotide sequences. As Williams (1989) highlights in a research paper, "polymorphisms are determined by the number and varying lengths of these DNA fragments.
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" These polymorphic regions are then amplified using primers, and through agarose gel electrophoresis, DNA bands are visualized with the aid of a dye and a UV trans-illuminator. The unique bands that emerge correspond to specific genotypes, rendering RFLP suitable for various applications such as forensics (DNA Fingerprinting), paternity testing, and identification of hereditary disease markers.
The strength of RFLP lies in its ability to link a particular genetic disease with a specific genotype, locating it on the gel based on the number of base pairs it encompasses. When a patient's DNA is subjected to RFLP analysis, and a matching locus is identified, it implies that the patient possesses the genetic marker for that particular disease.
This information can be pivotal in initiating preventative treatment. RFLP offers advantages such as a high level of reproducibility and reasonable accuracy. However, it is worth noting that it can be an expensive procedure, and consistency in results can sometimes be a challenge. Therefore, it is advisable to compare results when possible to ensure consistency.
Another potential issue with RFLP analysis, as mentioned in the National Research Council's book (1992), is band shifting during gel electrophoresis. Band shifting may occur due to variations in DNA concentrations among samples, leading to significant alterations in results.
In conclusion, the development of RFLP has been instrumental for molecular biologists due to its diverse applications in genetic analysis.
In this section, we outline the experimental procedures employed for the analysis of genetic traits using Restriction Fragment Length Polymorphism (RFLP). The following steps were carried out:
10 µl of Mastermix was dispensed into individual 0.5 ml Eppendorf tubes, with one tube assigned for each sample. These tubes were appropriately labeled with sample identification numbers and stored on ice.
The Mastermix consisted of:
It was essential to change the pipette tip between each sample to prevent cross-contamination. Subsequently, 5 µl of the DNA samples were added to their respective PCR tubes and immediately transferred to the thermal cycler. The cycling parameters were as follows:
| Temperature (⁰C) | Time |
|---|---|
| 94 | 4 minutes |
| 94 | 1 minute |
| 60 | 1 minute (x 30 cycles) |
| 72 | 1 minute |
| 72 | 10 minutes (final extension) |
| 4 | hold |
For MstII digestion of the 15 µl amplified reactions, 1.0 µl (5U) of MstII was added to each reaction, followed by incubation at 37⁰C for 1-2 hours. After the incubation period, the reaction was terminated by heating to 70⁰C for 5 minutes and then rapidly cooling on ice. Restriction digests were stored at 4⁰C.
A 2% (w/v) agarose gel was prepared by dissolving 2g of agarose (Sigma NA grade) in 98ml of sterile H2O. The agarose solution was heated until complete dissolution and then allowed to cool to 55⁰C before adding 2ml of 50x TAE buffer and 10µl of GelRed™ Nucleic acid stain. The resulting gel was poured into gel trays equipped with a 20µl slot comb. Care was taken to eliminate visible air bubbles before allowing the gel to set.
Each sample was prepared for loading onto the gel by adding 3µl of loading dye per 15µl sample. Additionally, 10µl of a 100 bp ladder was loaded into the first well, and 10µl of each sample was loaded into separate lanes.
The running buffer used for agarose gel electrophoresis of plasmid DNA samples was 1 x TAE buffer (0.04M Tris-HCl, 1mM EDTA, pH 8.0). Electrophoresis of samples was conducted at a constant voltage of 100V using the Pharmacia GNA-100 system or its equivalent, continuing until the dye front had migrated approximately 10cm. Subsequently, the gels were removed from the apparatus, and DNA bands were visualized using a UV transilluminator.
The molecular size of purified genomic DNA was estimated by comparing the relative mobility of the fragments with that of standard size markers run on the same gel. A 100bp ladder was used for basic analysis of the genotypes.
The results obtained from our analysis reveal important insights into the nature of the genetic trait under investigation. It is evident that the disease in question is not sex-linked, as it affects individuals of both sexes. Moreover, our findings suggest that the disease follows an autosomal recessive pattern, as not all individuals within the families are affected, and some may serve as carriers of the disease-causing gene. This indicates that for an individual to manifest the disease, they must inherit the disease-associated gene from both parents.
As we examine the results closely, it's crucial to address potential sources of error and limitations in our experimental approach. Firstly, the difficulty in reading P2, as shown in Figure 1, raises the possibility of human error playing a role in the results. Collaborative efforts involving different individuals in both the experimental procedure and result interpretation can introduce varying levels of precision.
Another notable observation is the presence of a single allele for most individuals in the families. This may be attributed to the complexities in gel application and interpretation. One potential improvement would have been to extend the electrophoresis time to enhance band clarity on the gel.
Although the pedigree analysis provides a useful framework for presenting the data, it is important to acknowledge the challenge in confidently assigning everyone to their correct family due to the difficulty in determining the alleles present in the gene.
In Family 1, there appears to be a prevalence of allele A3, yet not all individuals with this allele exhibit symptoms of the disease. This suggests the possibility of genetic crossing-over or other factors influencing disease expression. In contrast, in Family 2, allele A1 is linked to the disease, which is logical considering it is the allele most frequently cleaved by the primer, making it highly amenable to amplification by MstII.
In conclusion, our study demonstrates the utility of amplifying different regions of the gene using RFLP analysis for identifying genetic traits. The results indicate that alleles A1 and, likely, A3 are linked to the disease under investigation. This knowledge contributes to our understanding of the genetic basis of the disease and its inheritance pattern.
Genetic Trait Analysis Using RFLP - Lab Report. (2024, Jan 23). Retrieved from https://studymoose.com/document/genetic-trait-analysis-using-rflp-lab-report
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