Site-directed mutagenesis is a method used to construct amino acid changes in a protein in order to test the function of a specific amino acid. It is the way to study protein structure and function change the amino acid sequence of a protein by altering the DNA sequence of cloned gene4. A particular amino acid is very important in catalytic activity, ligand binding, protein folding or other function. Amino acid residue significance is tested by making conservative substitutions or by changing the amino acid to either alanine or glycine. Site-directed mutagenesis is also used to construct compensatory mutations, which are used to show the importance of specific interactions by making changes in both interacting partners. Each change individual destroy the interaction and combination of changes restores the interaction. These type of experiments are typically between two protein or between protein and nucleic acid. Used site-directed mutagenesis to investigate the interaction between hPol k and DNA1.
DNA polymerase kappa is a Y-family polymerase that use to bypass the damage DNA as translesion synthesis polymerase. In prostate cancer has reported that it has higher frequency of mutation in DNA polymerase kappa. 28% of patients have somatic mutations in POLK gene of prostate tumor and dominated by C- to –T transition2.
Approximately 30,000 DNA lesions occur per cell per day in mammalian cells. This damage to DNA can lead to cell death as well as disease such as cancer.
In this experiment, you will model the effects of mutations on the genetic code. Some mutations cause no structural or functional change to proteins while others can have devastating affects on an organism.
Most telomere are no more that the simple repetition of the DNA nucleotide sequence TTAGGG — two thymine bases, an adenine base, and three guanine bases. With a full length telomere containing more that 15,000 base pairs, telomeres can perform their functions of preventing chromosome from losing coding regions of the genome during replication for many replications before reaching what Dr. Jerry Shay describes as their “critical length” (Shay & Wright). When the telomeres of a cell reach this critical length and can no longer replicate without damaging the base pair sequences of the chromosomes themselves, programmed cell death occurs in the form of apoptosis. However, the length of a chromosome’s telomere is not only a linear function of erosion, but also a function of how often telomerase rebuilds the strands by adding the TTAGGG base pair sequence. It is this way to “cheat” the critical point that provides medicine with the possibility to extend our
Some chemicals can induce the modification of bases: Note: Conversion of C to U will result in a C:G to A:T mutation after replication
By helping to repair DNA, the BRCA2 protein plays a critical role in maintaining the stability of a cell 's genetic information. The cancer risk caused by BRCA2 mutations is inherited in a dominant fashion, even though usually only one mutated allele is directly inherited. This is because people with the mutation are likely to acquire a second mutation, leading to the dominant expression of the cancer. A mutated BRCA gene can be inherited from either parent. Because they are inherited from the parents, they are classified as hereditary or germline mutations. Because humans have a diploid genome, each cell has two copies of the gene (one from each biological parent). Typically only one copy contains a disabling, inherited mutation, so the affected person is heterozygous for the mutation. If the functional copy is harmed, however, then the cell is forced to use alternate DNA repair mechanisms, which are more error-prone. The loss of the functional copy is called loss of heterozygosity (LOH). Any resulting errors in DNA repair may result in cell death or a cancerous transformation of the cell.
To mutagenize human Kif5A (WT), we performed PCR-mediated site-directed mutagenesis to insert our mutation into the plasmid vector (shown to the right). Next, we performed a restriction enzyme digest with Dpn1, to digest the parental plasmid. The presence of the PCR product was confirmed by performing gel electrophoresis where the expected band of 6.8 kilobases was obtained. The remaining DNA was used in a bacterial transformation of DH5α to grow colonies. Liquid cultures were grown from overnight colonies and Minipreps were performed to purify the plasmid DNA. These plasmids were sent for sequencing, where the P278L mutation was confirmed.
mutation is phenylalanine 508 known as delta F508. Delta F508 is a deletion of 3 nucleotides
Cancer is a disease caused by an uncontrolled division of abnormal cells. The DNA sequence in cells can be changed as a result of copying errors during replication. If these changes whatever their cause are left uncorrected, both growing and non-growing somatic cells might gain many mutations that they could no longer function. The relevance of DNA damage and repair to the generation of cancer was obvious when it was recognized that everything that causes cancer also cause a change in the DNA sequence. Tumor suppressor genes are protective genes and normally they limit cell growth by monitoring the speed of cell division, repair mismatched DNA and control when a cell dies. When a tumor suppressor gene is mutated cells grow
A mutation is any type of alteration or change in DNA. There are many types of mutations that can occur. Depurination and deamination are common mutations that happen spontaneously. Depurination is a hydrolysis reaction that leads to the loss of purines in DNA. Deamination is also a hydrolysis reaction, and the cause of this reaction is an amino group gets detached. These types of mutations cause an alteration in the base sequence of amino acids and also effect the way a gene reads a protein. Another cause of DNA mutations may be environmental elements such as: chemicals or radiation. (pg. 567) A common chemical that is a mutagen is cigarettes and a example of
We know from the induced-fit model that the binding of the substrate (DNA) causes a conformational change in the active site of the enzyme. This model refers to a methylation reaction in which the cysteine needs to interact with cytosine of the DNA. Cytosine-5-methyltransferase transfers a methyl group from S-adenosyl-L-methionine to the C5 of cytosine. Cysteine residue attacks the cytosine causing the
Cancer known in medicine as a malignant neoplasm is one of the biggest killers worldwide. In 2007, cancer caused roughly 13% (7.9 million) of the planet’s deaths (Jemal, 2011). This will more greatly affect an aging society such as ours in years to come, and yet it is already the foremost cause of death in the developed world. The main reason cancer causes so many fatalities the body’s inability to mount an effective response to the failure of DNA replication within the body. This results in a mass
In prostate cancer, the second most common cause of death among men, lncRNAs such as PCA3, PCGEM1, SChLAP1, and
What is a Genetically Modified Organisms (GMOs)? GMOs is a plan or animal which through new biotechnology techniques, its genetic configuration has been changed. Foods from Genetically Engineered Organisms have been in our food supply for many decades now. With the rise in population and so many factors such as unpredictable weather; many pest and crops as well as animal diseases which hinder yields in agriculture. Scientist has tried to come up with some plants and animals that can withstand harsh environments. These foods are referred to as biotech foods or as many of us call them GMOs. This process gives rise to organisms which are better and they take less time to mature and are also a cost effective method. This is not to suggest that there is no disadvantages, in fact this is the reason why the government has come up with regulations to ensure that the products are safe for consumption and do not pose any health issues to the humans.
Cancer occurrs by the production of multiple mutations in a single cell that causes it to proliferate out of control. Cancer cells often different from their normal neighbors by a host of specific phenotypic changes, such as rapid division rate, invasion of new cellular territories, high metabolic rate, and altered shape. Some of those mutations may be transmitted from the parents through the germ line. Others arise de novo in the somatic cell lineage of a particular cell. Cancer-promoting mutations can be identified in a variety of ways. They can be cloned and studied to learn how they can be controlled.
DNA polymerase can have a particular function when it comes do different diseases or conditions. DNA polymerase has a specific function in the treatment of hepatitis B. The hepatitis B virus depends on a type of DNA polymerase called reverse transcriptase. When patients that are given a class of medication called analogue nucleosides. This medication specifically targets the DNA polymerase in the Hepatitis B Virus. In some Hepatitis B cases the DNA polymerase mutates in the presence of the treatment casing a resistance (Nguyen, Garcia, Trinh, Nguyen, Nguyen, Nguyen, & Levitt, 2009). It also can be seen in research to try to stop cancerous growth. DNA