The use of PCR and genetic approaches in biotechnology

(1) Define the following terms.

(a) Ecotilling
(b) An allelic series
(c) Functional redundancy
(d) Forward genetics and reverse genetics

(2) Of these following mutagens, transposons, T-DNAs, fast neutrons, ethylmethane sulfonate (EMS) or using RNAi;

(a) Which strategy(s) has a high probability of generating partial loss-of-function mutations?
(b) Which of these approaches has a high probability of resulting in a dominant mutant allele/transgene?
(c) Which strategy(s) has a high probability of resulting in a null mutation?
(d) Which of these approaches has a high probability of resulting in recessive mutant allele/transgene?
(e) Which strategy can you use to specifically down-regulate your gene of interest in a tissue specific manner?

(3) Sally has a T-DNA insertional mutant that results in the Arabidopsis plant having no wax on its stems but it still has wax on its leaves. She determines that the T-DNA has created a knockout mutation where it has inserted into the middle of the coding region of a gene she calls WAX3, which belongs to a small family of highly related enzymes known to control wax biosynthesis.

However to confirm that this waxless stem phenotype is caused by this WAX3 gene she creates an RNAi construct to the entire coding region of WAX3 linked to the strong 35S promoter that drives high expression in all plant tissues. With this construct she transformation Arabidopsis and obtains one transgenic plant. Although it has a waxless stem, the leaves are now waxless and the plant is bushier when compared to wild-type plants.

As she can detect no WAX3 transcript in the RNAi line she concludes that the RNAi approach has been more thorough in determining the function of WAX3 and that when it is completely eliminated by this approach it is involved in wax biosynthesis in leaves and stem as well as being involved in plant bushiness.

(a) Do you agree with her conclusion?
(b) What experiments could you do to refute or support her claims?

(4) Two duplicated genes (i.e tandem duplication) that encoded transcription factors, lie within a 5 kb region on chromosome 1 of Canola have been shown to be functionally redundant in controlling the biosynthesis of a toxic compound in canola seeds and hence the oil needs to be purified via a costly process. Therefore you want to completely eliminate the expression of both these transcription factors to produce plants for commercial use. Outline a strategy that could best achieve this outcome.

(5) A biotech company is aiming to modify seed oils in Canola, a close relative of Arabidopsis, but a species difficult to transform. These oils are produced specifically in the embryo during seed development. Currently there are no strong promoters that are known which express genes strongly in this tissue. Design a mutational strategy (simple flow diagram) that may lead to the identification of promoters or enhancers that could drive gene expression in embryo tissues that would be of use for expressing enzymes to result in modified seed oil compositions.

(6) Sally wanted to determine the biological function of her gene of interest, gene 1. Although the gene was very large, surprisingly when Sally went to order a T-DNA insertional mutant, there was no apparent T-DNA insertion within the coding region of this gene, whereas in much smaller flanking genes within this region had multiple T-DNA insertions.

(a) What could be the reason that no T-DNA insertions were found in coding region of this gene and could that suggest this gene could be involved in a particular biological process?

(b) Given that Sally is unable to obtain a T-DNA mutant for this gene, what other approaches could she take to investigate the function of this gene. (

(7) Jim was trying to determine the expression of the rat Adh gene in hearts of different species of rat. After grinding up the hearts, isolating total RNA with Trizol, he isolated mRNA with an oligodT column. Next he performed a DNaseI treatment (to prevent contamination from genomic DNA). He then carried out qRT-PCR (quantitative real-time PCR) as set out below in a 20 μL reaction. He used primers (that obeyed all the design rules for qRT-PCR, so assume there is nothing wrong with the primers) based on the sequence of his Adh genomic clone, and designed them to amplify sequences 3' of the Adh coding region so to ensure specificity, as there is a family of different Adh genes in rats that all have high sequence similarity in their coding regions. However after performing PCR, these primers failed to result in the production of any PCR product (assume there is nothing wrong with the PCR program, annealing temperature or the reagents used in the PCR reaction).

To try and understand why, he checked the quality of the mRNA on a 20% acylamide gel in TBE buffer, and from the resulting RNA smear he concluded that the mRNA had degraded and was of poor quality. Do you agree that this could be the reason? What other problems could there be with the protocol and what suggestions do you have that may enable Jim to obtain a PCR product. What controls can you suggest?

mRNA     5 ng/20 μL reaction
Primer    1 20 pmol
Primer    2 20 pmol
Mg2+     1.5 mM

1X PCR buffer (includes the syber green reagent, ions and pH buffer for Taq activity).
Taq        1.5 units/reaction

(8) A Biotech company is trying to determine the genes that are important in seed size and consequently make the assumption that seed size would be determined by regulatory genes such as transcription factors, rather than genes that code for enzymes, basic cellular machinery or structural enzymes. Hence they decide to focus on regulatory genes that are expressed in Arabidopsis seeds, and to determine what genes could potentially be involved in controlling seed development they use the commercially available Affymetrix chips ATH1 arrays that contain the majority of Arabidopsis genes. However a criticism of their approach is that many regulatory genes are usually in low abundance and may not be detected on a micro-array, so many important genes may be overlooked. What is a possible alternative strategy that could overcome this problem of low expression and list the pros and cons of this strategy when compared to their original micro-array approach? What strategy could they use to determine whether any of the seed expressed regulatory genes are in fact involved in determining seed size?

Alonso JM and Ecker JR (2006) Moving forward in reverse: genetic technologies to enable genome wide phenomic screens in Arabidopsis. Nature review genetics 7, 524- 536.

Gilchrist EJ, Haughn GW (2005) TILLING without a plough: a new method with applications for reverse genetics. Curr Opin Plant Biol. 8, 211-215.

Comai L and Henikoff (2006) Tilling: practical single-nucleotide mutation discovery. Plant J, 45, 684-694.

An G, Jeong DH, Jung KH, Lee S. (2005) Reverse genetic approaches for functional genomics of rice.Plant Mol Biol. 59, 111-123.

Li X, Zhang Y. (2002) Reverse genetics by fast neutron mutagenesis in higher plants.Funct Integr Genomics. 2, 254-258.

Krysan PJ, Young JC, Sussman MR. (1999) T-DNA as an insertional mutagen in Arabidopsis.Plant Cell. 1999 11, 2283-90.

Waterhouse and Helliwell (2003) Exploring plant genomes by RNA induced silencing. Nature Review Genetics 4, 29-38.

Feng & Mundy (2006) Journal of Integrative Plant Biology 48, 5-14

Czechowski et al., (2004) Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reals novel root- and shoot-specific genes. The Plant Journal 38, 366-379.