T2

Question 1

Why was Genetic Engineering or Recombinant DNA technology developed?

Answer 1

It was as a result of several decades of basic research on DNA, RNA and viruses, that made it possible to develop technology that could precisely change in designed
ways the genetic endowment of organisms.

Question 2

What are type II restriction endonucleases and who were their discoverers?

Answer 2

Restriction endonucleases are enzymes that recognize specific nucleotide sequences and cut DNA at a specific section (restriction size). Specifically, the type II
enzymes always cut the DNA at equivalent points, near or far away from the recognition site. Most require Magnesium.

They were discovered by Arber, Nathans
and Smith. (1978)

Question 3

Who made the first recombinant plasmid?

Answer 3

Stanley Cohen

Question 4

Why are the hydrogen bonds between proteins and DNA stronger than those formed with water?

Question 5

Why do restriction endonucleases recognize palindromic sites?

Answer 5

Because those enzymes are homodimers, each monomer recognizes 3 bp from opposite sides of the double helix. So the site must be palindromic to allow both monomers to bind without mutual interferences.

Question 6

What is the role of DNA ligase?

Answer 6

Catalyzing phosphodiester linkages between nucleotides at the 5’ end of one strand of DNA and nucleotides at the 3’ end of the other strand.

Question 7

Why reverse transcriptase is important and who discovered it?

Answer 7

As RNAs only have one single strand by using reverse transcriptase it is able to create the complementary strand, making a double strand. So, these enzymes make RNA accessible to genetic engineering. These only exist in animal
retroviru.

Discovered by Renato Dulbecco, Howard Temin and David
Baltimore. The last one forgot to patent the enzyme.

Question 8

What plasmid was constructed by Joachim Messing?

Answer 8

Joachim Messing designed the plasmids that produce colonies with color white (insert) or blue (no insert) when transformed into bacteria expressing the beta fragment of beta-galactosidase.

Question 9

Who developed the Gateway system and what does it consist of?

Answer 9

Gateway system was developed by James L. Hartley, it consists of cloning by recombination instead of cutting and paste, thanks to the λ phage recombination sites and recombinases.

Question 10

What are the uses of hybridization?

Answer 10

Hybridizations are used for base-pair language: probes, primer extension, Southern, northern, microarrays…

Question 11

Why is the solid-phase synthesis of oligonucleotides important?

Answer 11

This chemical tool is crucial in Genetic Engineering for synthesizing primers to sequence DNA and also for flanking primers in PCR (DNA polymerases require primers).

Question 12

Frederick Sanger method of sequencing.

Answer 12

Sanger sequencing is a method of DNA sequencing that involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides byDNA polymerase during in vitro DNA replication. These dideoxynucleotides are chain-terminating nucleotides that lack a 3’-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA when a modified ddNTP is incorporated. The ddNTPs may be radioactively or fluorescently labeled for detection in automated sequencing machines. There are four fluorescent dideoxynucleotides of different colors, one for each base

Question 13

When was the sequence of one human genome completed?

Answer 13

The sequence of the human genome was completed in 2003 sizing 3.000Mb and with 21.000 genes coded

Question 14

Does the size of genomes correlate with the evolutionary level of an organism?

Answer 14

The size of genomes and the number of genes does not correlate with the evolutionary level of an organism. For example, plants are the organisms with greater genomes (e.g: wheat has 17 000 Mb and an estimation of 200.000 genes) but humans are more complex organisms

Question 15

Describe one method of Next Generation Sequencing

Answer 15

In the reversible terminator method, the four nucleotides are added to the templateDNA, with each base tagged with a
unique fluorescent label and a reversibly-blocked
3’ end. The blocked end ensures that only one phospho- diester linkage will be formed. Once the nucleotide is incorporated into the growing strand, it is identified by
its fluorescent tag, the blocking agent is removed, and the process is repeated. In pyrosequencing, nucleotides are added to the template DNA, one at a time in a
defined order. One of the nucleotides will be incorporated into the growing strand, releasing a pyrophosphate which is detected by coupling the formation of pyrophosphate with the production of light by the sequential action of the enzymes ATP sulfurylase and luciferase:

The protocol for ion semiconductor sequencing is similar to pyrosequencing except that nucleotide incorporation is detected by sensitively measuring the very small changes in pH of the reaction mixture due to the release of proton upon nucleotide incorporation.

Question 16

Principle of nanopore sequencing

Answer 16

In this method chains of single strand DNA or RNA pass through a channel made by the protein alpha-hemolysin inserted in a membrane surface. This is driven by an
electrical potential and every nucleotide of the sequence produces a different signal of ion current. It is the most promising method for the future.

Question 17

How site-directed mutagenesis is made

Answer 17

Site-directed mutagenesis causes mutations called point mutations since only one base is altered.
These mutations can be made if we have a plasmid containing the gene or cDNA for the desired protein and if we know the base sequence around the site to be altered.
To introduce this mutation into our plasmid, we prepare a primer that is complementary to the region of the gene except that it contains one different nucleotide.
The two strands of the plasmid are separated, and the primer is then annealed to the complementary strand. The mismatch of 1 of 15 base pairs is tolerable if the
annealing is carried out at an appropriate temperature. After annealing to the complementary strand, the primer is elongated by DNA polymerase, and the double-stranded circle is closed by adding DNA ligase.
Subsequent replication of this duplex yields two kinds of progeny plasmid, half with the original sequence and half with the mutant sequence. Expression of the plasmid
containing the new sequence will produce a protein with the desired substitution of an aminoacid due to the base change.

Question 18

Types of molecular markers

Answer 18

SNPs (1bp), Satelytes(20-100bp), Minisatelytes (5-20bp) and microsatelytes (2-4bp)

Question 19

What is the use of quantitative PCR and how is it done?

Answer 19

In a quantitative PCR, the accumulation of amplification products is measured as the
reaction progresses, by the inclusion of a fluorescent reporter molecule or DNA intercalating dyes in each reaction. The increase in the fluorescence is related with an increase in the amount of product. The amplification detection of a quantitative PCR is carried out in a closed-tube format after each cycle, which eliminates the need for post-PCR manipulation, such as gel electrophoresis and significantly
reduces the risk of cross contamination. Quantitative PCR has multiple applications such as gene expression analysis, the
detection of genetically modified organisms in food and cancer phenotyping.

Question 20

What is the goal of Genetic transformation?

Answer 20

The final goal of genetic engineering is genetic modification to test hypotheses and to generate biotechnological applications.

Question 21

How are transgenic mice done?

Answer 21

Cloned plasmid DNA is being microinjected with a micropipette into the male pronucleus of a fertilized egg of a mouse. This DNA will join by random to the chromosome.

Question 22

How is human insulin done?

Answer 22

The pancreas synthesizes the mammalian proinsulin mRNA, then thanks to the
reverse transcriptase, a complementary strand is synthesized, obtaining the
proinsulin cDNA. Joining this cDNA to a plasmid we obtain a recombinant
plasmid, which will be inserted into an E. Coli. This gene has the information to
synthesize the mRNA involved in the synthesis of the proinsulin. That is why when
the plasmid is inside the E.Coli, this will produce the corresponding mRNA and … the
proinsulin.

Question 23

How are genes disrupted by homologous recombination and what is learned from it?

Answer 23

Similar DNA molecules align, and nucleotide sequences are exchanged between these two molecules (crossover). This exchanging of DNA is an important source of the genomic variation seen among offspring.

1. (A) A mutated version of the gene to be disrupted is constructed, maintaining some regions of homology with the normal gene (red).
2. When the foreign mutated gene is introduced into an embryonic stem cell, (B) recombination takes place at regions of homology
3. and (C) the normal (targeted) gene is replaced, or “knocked out,” by the foreign gene.
   The cell is inserted into embryos, and mice lacking the gene (knockout mice) are produced.

Question 24

How transgenic plants are made and who were the discoverers?

Answer 24

Transgenic plants are formed by inserting a desirable stretch of DNA sequence into plant cells, and then proliferating it using plant tissue techniques. Genetic engineering techniques are used to change the DNA of transgenic plants.
The discoverers of plant genetic transformation are Marc Van Montagu and Jeff Schell, but the Mexican scientist Luis Herrera-Estrella also participated in the discovery.

Question 25

What are the Ti plasmids?

Answer 25

It is an extrachromosomal circular molecule of DNA found commonly in the plant pathogen Agrobacterium tumefaciens. It enables the bacterium to infect plant cells and produce a tumor.

Question 26

How are cells transformed by electroporation?

Answer 26

The application of intense electric fields makes membranes and cell walls transiently permeable to DNA, allowing gene
transfer and the generation of genetically modified cells.

Question 27

How have the methods of Genetic Engineering changed at present?

Answer 27

The hybridization (northern and southern) used in the past has been replaced by massive sequencing (RNAseq, DNAseq, qRT-PCR, digital PCR). From now on, recombinases are going to be used instead of restriction endonucleases and ligases.
Gene libraries and hybridization screening have also changed to PCR, ordered in Genome Databases. Finally, TILLING (Targeting Induced Local Lesions in Genomes), Genetic transformation and RNAi have been substituted by
CRISPR-Cas9.

Question 28

How does RNA silencing work?

Answer 28

The RNA silencing or the RNAi pathway is based on two
steps, each involving ribonuclease enzymes. In the first
step, the trigger RNA (dsRNA or miRNA) is processed into
a short interfering RNA (siRNA) by the RNase called Dicer.
In the second step, siRNA is loaded into the effector
complex RISC (RNA-induced silencing complex). The
siRNA is unwound during RISC assembly and the
single-stranded RNA hybridizes with mRNA target. Gene
silencing is a result of nucleolytic degradation of the
targeted mRNA by an enzyme of the RISC.

Question 29

How does CRISPR-Cas9 genome editing work and who discovered it?

Answer 29

The RNA-guided DNA endonuclease works by making a directed genomic double strand cut that will open the way for genome editing by homologous recombination.
The CRISPR system was discovered by Francisco Martínez but the novel for the discovery was given to Emmanuel Charpentier and Jennifer Doudna, who developed
the applications

Question 30

Why the guide RNAs in both RNA silencing and CRISPR-Cas9 need to be 20 nucleotides long?. Compare the different methods of genetic modification.

Answer 30

Because recognition of 20 bp is needed to target a site present once in large genomes or transcriptomes and normal DNA binding proteins cannot recognize so many bp
1/420 = 1/1.1 x 1012 ≈ 1 site/ 106 Mb (big wheat genome has 17000 Mb)

Question 31

Why are DNA polymerases of some archaea specially useful for PCR?

Answer 31

Because some archaea live at high temperature and have enzymes such as DNA polymerase that are thermoresistant