Lab Skills in Molecular Biology and Genetics

Syllabus

During this course, the students set biological questions, design appropriate experimental procedures, apply the corresponding experimental methods and finally answer the initial question. The course is divided into two sections.
In the first section, the molecular basis of bitter taste perception is investigated using the synthetic, non-toxic chemical bitter substance phenylthiocarbamide (PTC) as one of a series of natural and synthetic bitter substances recognized by the same taste receptor, TAS2R38. This gene has a common polymorphism that mainly determines the taste ability of PTC. Based on these, phenotypic and genetic testing is performed on students who wish to participate in the research (the remaining students use data and samples from anonymous volunteers). To identify this specific polymorphism, polymerase chain reaction (PCR) is performed in combination with restriction fragment length polymorphism (RFLP) analysis. In order to understand the further possibilities of utilizing the amplification of a DNA segment by PCR (both in the context of genotyping and in further applications), cloning of the PCR product and mapping follows.
In detail, the following experimental procedures are carried out:

• Taste test with PTC and negative control paper strips. Recording of the bitter taste perception in the students and family members and creation of a pedigree.
• Isolation of genomic DNA from swab cells. Quantitative and qualitative control of DNA by spectrophotometry and agarose gel electrophoresis.
• Polymerase chain reaction (PCR) to amplify a segment of the TAS2R38 gene containing the variant in question.
• Restriction fragment length polymorphism (RFLP) analysis targeting the specific variant and determination of the genotype by agarose gel electrophoresis.
• Ligation reaction of the PCR product with a plasmid cloning vector.
• Preparation of competent coli cells and transformation of the ligation reaction product.
• Selection and growth of colonies based on galactosidase staining and isolation of plasmid DNA on a small scale.
• Mapping of recombinant plasmids.

The second section of the course examines the genetic linkage and the estimated genetic distance between three autosomal genes of the Mediterranean fly (Ceratitis capitata) that determine a) the body colour in the pupa stage (white wp/brown wp+), b) the eye color in adults (white w/green-red w+). The genetic linkage and distance between them will be determined by crossing suitable strains and counting the offspring that have recombinant and non-recombinant phenotypes. In particular, the following experimental procedures will be carried out:

• Familiarization with the handling of fly strains (study of developmental stages, discrimination of males/females, observation of mutant strains, nutrition, collection of eggs and pupas).
• Crossing of two purebred strains (P generation) with dominant and recessive phenotypes respectively for both genes.
• Crossing of a female offspring heterozygous for both genes with a male individual carrying the recessive traits (control cross) as well as a reverse cross regarding the sexes (F1 generation).
• Counting of offspring with recombinant and non-recombinant phenotypes in the two alternative crosses and estimation of genetic distances between the genes. Comparative analysis of the results between student groups.

Student Performance Evaluation

Student performance is evaluated based on the laboratory report (20%), the tests carried out at the beginning of the laboratory exercises or tutorials (20%) and the written examination during the examination period (60%).
The written examination includes:
• experimental procedure design and critical thinking questions (80%).
• experimental calculations, knowledge and experimental applications questions (20%).

Suggested Bibliography

• Introduction to Genetic Analysis, 12th Edition by Anthony Griffiths, John Doebley, Catherine Peichel, David Wassarman, W.H. Freeman & Company, 2020.

• iGenetics: A Molecular Approach (3rd Edition) by Russell, Peter J. published by Benjamin Cummings (2009)

Teaching Material / E-class

https://eclass.uth.gr/courses/BIO_U_205/