Genetic engineering techniques

What is the polymerase chain reaction (PCR) and what is it used for? Name two practical situations in which amplifying DNA is essential.

The diagram shows a PCR cycle with its three numbered steps and the temperature of each step. Explain what happens in each step and reason why each requires its specific temperature.

To amplify a target double-stranded DNA fragment by PCR, the sequence of the ends of one of its strands (read in the 5' $\rightarrow$ 3' direction) is known:

5'-ATGCCGTAG... [target fragment of $600\ \text{bp}$] ...ACGTTCGAT-3'

1. a) Design the sequence and polarity (5' $\rightarrow$ 3') of the two DNA primers (of 9 nucleotides each) needed to amplify this region. Justify why both primers must have different sequences and be oriented in convergent directions.
2. b) A student programs the thermal cycler with the following cycles: $60\ \text{°C}$ (annealing) and $72\ \text{°C}$ (extension), intentionally omitting the step at $95\ \text{°C}$. After 30 cycles, they analyze the sample by electrophoresis and observe no bands. Justify at the molecular level the cause of the failure.
3. c) Assuming an initial 2 double-stranded DNA molecules and a theoretical reaction efficiency of $100\ \%$, deduce how many double-stranded molecules containing exclusively the exact length delimited by the primers will have been generated upon completion of the third PCR cycle. Justify your answer by reasoning step by step what happens in each cycle.

In a laboratory, researchers intend to amplify a human gene using PCR, but by mistake a technician uses purified DNA polymerase I from Escherichia coli instead of the standard Taq polymerase from Thermus aquaticus.

1. a) If the standard thermal program is applied ($95\ \text{°C}$ for $30\ \text{s}$, $55\ \text{°C}$ for $30\ \text{s}$, and $72\ \text{°C}$ for $1\ \text{min}$, repeated 30 times), predict what result will be obtained in agarose gel electrophoresis and molecularly justify what happens to the E. coli enzyme during the first cycle.
2. b) In the early days of the technique, before the isolation of thermophilic polymerases, PCR was performed with E. coli DNA polymerase I. Design the modification to the experimental protocol that researchers had to carry out in each of the 30 cycles to achieve amplification, and explain what practical drawbacks this posed.
3. c) Taq polymerase lacks 3' $\rightarrow$ 5' exonuclease activity, unlike other thermostable polymerases such as Pfu. Justify which of the two enzymes is more suitable for amplifying a gene for therapeutic purposes where sequence errors cannot be tolerated, explaining the function of said enzymatic activity.

What are restriction enzymes, from which organisms are they obtained, and what is their function in genetic engineering?

What is a transgenic organism? Give an example and outline, in general terms, how it is produced.

Put the steps required to clone a human gene in bacteria in order and justify the sequence: introduce the recombinant plasmid into the bacterium, cut the gene and the plasmid with the same restriction enzyme, join the gene to the plasmid using DNA ligase, select and multiply the transformed bacteria.

A research group aims to synthesize the peptide hormone insulin in the bacterium Escherichia coli. To do this, they directly isolate the insulin gene from the nuclear DNA of human pancreatic cells and insert it into a bacterial plasmid alongside an efficient E. coli promoter.

1. a) After introducing the plasmid into the bacteria, they find that an aberrant, inactive polypeptide of incorrect size is synthesized. Explain why eukaryotic genomic DNA cannot be properly expressed in a bacterium, referencing gene structure and RNA processing machinery.
2. b) Design the biotechnological strategy to solve this problem: indicate which molecule you should isolate as the initial template, what type of specific synthetic primer you would use taking advantage of a post-transcriptional modification of that molecule, and which enzyme of viral origin you will use to synthesize the first strand of complementary DNA (cDNA).
3. c) When comparing the sequence of the obtained cDNA with that of the initial nuclear gene, it is observed that the cDNA is much shorter and lacks the original human promoter sequence. Explain why this happens and justify how the bacterium is made to transcribe this cDNA once cloned into the plasmid.

CRISPR-Cas9 allows genome editing with high precision. Explain simply what role guide RNA plays and what role the Cas9 protein plays in this technique.