Biotechnology

Question 1

In situ hybridization

Answer 1

In situ hybridization allows us to determine location of a DNA molecule (typically in a patient tissue section or chromosomes). For in situ hybridization, recombinant DNA that is known to have a sequence that is complementary to a specific DNA sequence of interest is synthesized and a label is added to this nucleotide sequence (the labeled recombinant DNA is usually referred to as a probe) – we use in situ hybridization when we want to find the location of a DNA sequence/gene within a tissue or chromosome. We can also use it to determine how many copies of a gene a chromosome has (in situ hybridization can be used to identify gene deletions and amplications).

• Detect the presence and chromosomal location of a reciprocal translocation.

Question 2

PCR

Answer 2

• PCR is able to detect the expression of DNA sequences that are expressed at very low levels (important for detecting the presence of low level infections in patients).
• Can be used to determine if a CMV viral infection has been irradicated

Question 3

PCR followed by sequencing

Answer 3

• Sometimes PCR products are sequenced to determine whether a genetic alteration (mutation) is present.
• Ex. used to determine whether a missense mutation has occured in a gene following exposure to a mutagen
• There are several options, including the classic method of dideoxy DNA sequencing. A DNA primer and DNA polymerase are required for dideoxy DNA sequencing as well as normal and labelled chain terminating nucleotides. Introduction of a labelled chain terminating nucleotide into a growing chain causes chain termination – as ‘normal’ as well as chain-terminating nucleotides are present in the reaction many different chains of different length are generated. Determination of the relative length and the terminal nucleotide of each of these chains can be used to determine DNA sequence.

Question 4

RNA Interference

Answer 4

• Determine whether a protein is essential for cell function
• this causes the degradation of a specific mRNA or prevents the mRNA interacting with the ribosome (either of these mechanisms prevents the protein being made from the mRNA = we can determine the impact of no expression of the protein and cell function/physiology).

Question 5

Introduction ( transfection) of a recombinant DNA into a cell line

Answer 5

• If we want to determine the impact of a genetic alteration cell function, we can introduce recombinant DNA (usually plasmids) into mammalian cell lines where it will be transcribed and translated = the protein is made in the mammalian cell and we can determine its impact on cell function/physiology. We can assess the function of a gene through inhibiting its expression.
• Determine the effect of a missense mutation on cell function

Question 6

knock-out

Answer 6

so no active gene is present

Question 7

knock-in

Answer 7

• addd additional copies of a gene
• use a knock in mouse model to investigate the impact of a missense mutation on organ function