Tutor profile: Lokesh B.
How many different types of gametes are possible with following genotype AbDdEe
Eight types of gametes is possible. Type of gametes can be calculated by 2n (2 the power n), where n is the number of gene in heterozygous condition
Subject: Basic Chemistry
Suppose you are involved in analyzing the gene expression pattern of certain genes in the breast cancer cell line. Using RT-PCR and western blot analysis, you demonstrated that gene X is up-regulated in all breast cancer cells as compared to the non-cancerous cell line. Based on RT-PCR and western blot data, your supervisor hypothesized that gene X is an oncogene. You have given the assignment to validate this hypothesis. What would be your experimental design to validate this hypothesis?
Answer: All cancer cells share certain traits such as uncontrolled proliferation and the ability to metastasize. These traits are governed by the oncogene. The oncogenic nature of gene X can be examined by following approaches. Gene X will be cloned and expressed in a non-cancerous cell line. If gene X is Oncogenic, a non-cancerous cell will begin to behave like cancerous cell and exhibits uncontrolled cell growth and metastasis. Uncontrolled cell growth can be examined by MTT assay, whereas metastasis ability can be examined by the wound-healing experiment. Another approach to demonstrate the oncogenic nature of gene X is as follows. Gene X will be knockdown in cancer cells using the anti-sense RNA or CRISPR-Cas9 method. If gene X is Oncogenic, cancer cell would lose the ability for uncontrolled proliferation or the ability to metastasize.
Let's assume you received two DNA samples: DNA A and DNA B. One of DNA contains promoter sequences. Design an experiment that allows you to find out which DNA samples contain the promoter sequences. Assume you have access to all the required reagents (like primers, polymerase enzyme, dNTP, plasmids, etc.) and resources to execute the experiments.
Luciferase reporter assay is the most accepted tool to identify and characterized the promoter sequence. Both DNA samples will be clone in pGL3 Luciferase reporter plasmid, individually. A reporter plasmid containing DNA A named as pGL3-A whereas, reporter plasmid containing DNA B named as pGL3-B. Mammalian cell line will be grown in 3 wells of six-well plate up to 70%–80% confluence. One well will be transfected with plasmid pGL3-A, the second well will be transfected with plasmid pGL3-B and third well will be transfected with vector alone pGL3 (served as a negative control). After incubation for a desired time, luciferase activity will be measured in each transfected sample. A DNA sample that contains the promoter sequence would show luciferase activity whereas a DNA sequence that does not contain promoter sequence would NOT show luciferase activity. For example, if we detect luciferase activity in a sample transfected with pGL3-A plasmid. It implies that DNA A contains a promoter sequence.
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