GENE MANIPULATION BIOLOGY UNIT 4 SAC

Isolated plasmids can replicate.

False- A plasmid must be within a host cell to replicate.

One person could have 10 different alleles at one STR locus.

False- One person could have two different alleles at one STR locus.

All recombinant proteins are replacements for proteins that were extracted from mammalian, including human, tissues in the past.

"False- Some of these recombinant proteins, such as Humulin, replaced products that were previously extracted from tissues of other species. Other recombinant proteins, such as human growth hormone (rec hGH), replaced products that were extracted from tissues of dead people (cadavers). Other recombinant proteins replaced products that were extracted from tissues of living people, such as follicle stimulating hormone (FSH), which was previously extracted from the urine of post-menopausal women, and hepatitis B vaccine, which was previously based on antibodies extracted from the plasma of people with chronic hepatitis infection. Finally, other recombinant proteins are new products that have only ever been produced through the expression of cloned genes.

What is meant by the phrase 'denature — bind primers — extend primers'?

"These three steps are part of the process of the polymerase chain reaction. The polymerase chain reaction depends on the enzyme DNA polymerase to amplify or make multiple copies of a sample of DNA. 1. *Denature*: the DNA sample is denatured by heating so that it dissociates to single strands (94 °C for two minutes). 2. *Anneal*: Bind primers: short segments of single-stranded DNA, known as primers, are added; these primers pair with regions at either end of the DNA region of interest (55 °C for two minutes). 3. *Extension*: Extend primers: the polymerase enzyme uses the primers as a starting point and extends them so that two complete double strands are formed. For this step, a supply of nucleotides must be available.

ELECTROPHORESIS

A lab technique using electrically charged gel to separate DNA fragments.

Identify two procedures that can increase the chance of uptake of plasmids by bacteria.

1. One technique is termed electroporation. In this method, cells are briefly placed in an electric field that shocks them and appears to create holes in their plasma membranes so that plasmid entry is facilitated. 2. Another method is to heat shock the bacterial cells by suspending them in an ice-cold salt solution, and then transferring them to an environment of 42 °C for less than one minute. This treatment appears to increase the fluidity of the plasma membranes of the bacterial cells and increases the chance of uptake of plasmids by the bacterial cells.

GENE OF INTEREST

A gene for a desired trait; the one being added to a plasmid.

CLONE

A genetically identical organism created asexually from the cells of another organism

HOST CELL

A host cell contains foreign DNA and can produce the protein coded by the foreign DNA.

Having obtained a copy, describe one means by which the scientist can obtain millions of copies of the gene.

A scientist can obtain millions of copies of a gene using the polymerase chain reaction (PCR). In contrast to gene cloning, which occurs inside cells, PCR can occur in a test tube.

DNA FINGERPRINT

A unique pattern of bands produced by electrophoresis.

Identify one capability provided by CRISPR-Cas9 technology that was not possible with earlier gene therapy techniques.

An efficient and reliable technique for making precise and targeted changes to the genome of living cells was made possible with CRISPR-Cas9 technology. This has not been possible with earlier gene therapy techniques. Early gene therapy techniques added DNA segments to genomes but at unpredictable locations.

LIGASE

An enzyme used to bond DNA back together after splicing when making recombinant DNA (like glue)

GENETICALLY MODIFIED ORGANISM (GMO)

Another term used for crops that have been genetically engineered.

PLASMID

Circular DNA from bacteria used in making recombinant DNA

In bacterial transformation, bacteria take up foreign ________.

DNA

Population screening is for genetic disorders that are incurable.

False- Population screening is used to achieve early detection or exclusion of inherited disorders. An effective intervention or treatment for the disorder must exist to address the condition in those people found to have the disorder.

Presymptomatic testing involves identifying the carrier status of an individual.

False- Presymptomatic testing is used to detect gene mutations associated with disorders that appear after birth, often later in life.

A minisatellite is also known as a short tandem repeat.

False- Short tandem repeats or STRs (microsatellites) are hypervariable regions of chromosomes where sequences of just two to five base pairs are repeated over and over. Minisatellites are chromosomal regions where sequences of 9 to 80 base pairs are repeated tens or hundreds of times.

In a mixture of plasmids and foreign DNA, both with matching sticky ends, it would be expected that the plasmids would all join with the foreign DNA fragments.

False- The plasmids may re-join with their own sticky ends and not join to a plasmid.

The current technique of identification by DNA is known as DNA fingerprinting.

False- Today, individuals can be identified through a technique known as DNA profiling.

Host cells used in gene cloning can only be bacterial cells.

False- Yeast cells and other eukaryotic cells can also be used as host cells. Yeast cells or hamster ovary cells must be used when the final functional protein is a glycoprotein.

When CRISPR is used to add DNA to a genome, it is added in a random location.

False. CRISPR technology inserts a specially designed DNA sequence into a precise location in the genome.

CRISPR technology can only be applied to certain species.

False. CRISPR technology is now being put to use in editing faulty genes and in silencing genes in many plants and animals.

When bacterial cells and plasmids are mixed, all bacterial cells will be transformed by taking up the plasmids.

False. Cut plasmids can reseal themselves so that they are not recombinant plasmids.

A restriction enzyme that can act on human DNA would not be able to act on mouse DNA.

False. DNA has an identical composition in all living organisms and so an enzyme that can act on DNA from one source can also act on DNA from any other source.

Electrophoresis separates DNA fragments according to their different charges.

False. Electrophoresis separates DNA fragments according to their lengths (that is, their sizes).

Plasmids are the main chromosome of a bacterial cell.

False. Plasmids are small circular pieces of double-stranded DNA within a bacterial cell.

The reverse transcriptase process can be used to produce cDNA copies of noncoding DNA.

False. Reverse transcriptase enzyme can use an mRNA template to build a single strand of complementary DNA (cDNA). The mRNA is produced using a coding section of DNA.

In electrophoresis, DNA fragments travel from slots at the origin of a gel to the cathode.

False. The cathode is negatively charged. DNA has a negative charge and will move towards the positively charged anode.

Restriction enzymes cut DNA molecules at random sites along their length.

False: Cuts made by restriction enzymes do not occur randomly but at specific recognition sites along a double-stranded DNA molecule.

The restriction enzyme Aha I produces DNA fragments with sticky ends.

False: The restriction enzyme Aha I produces fragments with blunt ends.

TRANSGENIC

Having a mix of genes from two different species, combining their qualities.

CRISPR-Cas9 technology allows for gene editing. An issue that is the subject of debate is: Should gene editing be allowed on human cells, such as eggs, sperm and embryos, whose DNA can pass to future generations? Or, should gene editing be restricted to somatic cells where that DNA is not passed to the next generation?

Like any technology, CRISPR will be identified as good or bad depending on the uses to which it is put and their immediate and longer term consequences. As the technology is applied more widely, more unknowns will emerge. Aspects of the use of CRISPR technology raise ethical considerations that will be widely debated in both the scientific and public communities.

RECOMBINANT DNA

Made by adding desired genes into bacterial plasmid DNA.

Describe one means by which a scientist can obtain a copy of a gene.

Methods by which a scientist can obtain a copy of a gene include: 1. Cutting out and isolating a gene. This method involves the use of a gene probe and is limited to genes for which a gene probe is available. 2. Making a gene by chemical synthesis. Chemical synthesis is limited to genes or parts of genes about 800 nucleotides long. The nucleotide sequence of the gene must be known. 3. Making a copy of a gene using an mRNA template. Both an mRNA template and reverse transcriptase enzyme are necessary for this reaction.

STICKY ENDS

Opened unpaired base pairs, ready to be matched with complimentary bases.

RECOMBINANT PROTEIN

Recombinant proteins are proteins that are expressed by recombinant DNA present in an organism.

If an ampicillin-sensitive cell takes up a plasmid with an AmpR selectable marker, that cell will become ampicillin resistant.

TRUE

why the blue colour fails to appear in colonies of cells derived from cells possessing recombinant plasmids, where those plasmids have a lacZ screening marker that includes the recognition site for the restriction enzyme used to cut plasmids.

The DNA of the lacZ screening marker encodes the enzyme galactosidase, which converts a particular substrate to a blue-coloured product. If foreign DNA is incorporated at the recognition site in the lacZ marker, the DNA coding sequence of lacZ is disrupted so that no functional enzyme is formed and the blue substrate cannot be formed.

GENOME

The DNA sequence for an entire organism.

List two advantages of DNA profiling as compared to DNA fingerprinting.

The amount of DNA needed for DNA profiling is very small because DNA can be amplified through the polymerase chain reaction (PCR). DNA profiling is carried out in a much shorter time — hours rather than days.

GENETIC ENGINEERING

The process of physically or chemically changing DNA for a specific purpose.

why the same recognition sites must be present on the ends of the gene of interest and at a known site in the selected plasmid

The same recognition site will allow the same restriction enzyme to be used to cut both the plasmid and the gene of interest. This will produce matching sticky ends (or matching blunt ends) on the plasmids and the gene of interest. Many plasmids will join with the DNA of the gene of interest to form recombinant plasmids (that is, with DNA from different sources).

BIOETHICS

The study of the morals of biology, debating the line between right and wrong.

One role of a plasmid in gene cloning is to incorporate a gene of interest into its structure for transfer to a host cell.

True - A plasmid must be able to incorporate the gene of interest into its structure. This is often achieved by cutting the plasmid and the gene with the same restriction enzyme.

"The first recombinant protein to be produced for clinical use was insulin.

True -The first recombinant protein to be developed and made available commercially was Humulin (the commercial name for insulin).

The past source of a vaccine for hepatitis B made use of antibodies from the plasma of people with a chronic hepatitis B infection.

True -The hepatitis B vaccine was previously based on antibodies extracted from the plasma of people with chronic hepatitis infection.

The length of the repeated unit in an STR is from two to five base pairs.

True- STRs are hypervariable regions of chromosomes where sequences of just two to five base pairs are repeated over and over.

A gene of interest to be cloned can be produced by chemical synthesis.

True- The DNA of the gene of interest can be obtained, either by making it using a DNA synthesiser, or by using its mRNA template to make a cDNA copy of the gene.

One role of a recombinant plasmid is to clone the gene of interest after the plasmid has been taken up by a host cell.

True- The plasmid must carry the gene of interest into a living cell and, once there, the plasmid must replicate itself and produce multiple copies of (or clone) the inserted gene.

Chromosomal DNA gives more precise identification than mtDNA.

True- mtDNA identification is less precise because individuals from the same maternal line have identical mtDNA profiles.

Bacteria that are ampicillin sensitive will be killed by exposure to this antibiotic.

True.

Plasmids are self-replicating entities.

True. All plasmids are self-replicating because they contain an origin of replication (ORI), which is a specific DNA base sequence where DNA replication of a plasmid begins.

If ampicillin-sensitive bacteria take up plasmids that contain AmpR, these bacteria will be transformed and will become ampicillin resistant.

True. Bacteria that have taken up the plasmid with the antibiotic-resistant gene are unaffected by the antibiotic.

CRISPR can be used to correct faulty genes.

True. CRISPR technology can not only add DNA but can also edit, modify, or disable or delete DNA from the genome.

The scissors in CRISPR technology consist of a bacterial endonuclease enzyme.

True. Cas9 is an endonuclease that can cut double-stranded DNA from within the DNA.

In electrophoresis, shorter fragments of DNA travel further than longer fragments.

True. In a given time, the shorter fragments of DNA will move further from the well than larger segments of DNA.

The foreign DNA in a recombinant plasmid could come from mice or men.

True. In recombinant plasmids, the foreign DNA that they incorporate can come from any source: it may be a human gene, a plant gene, a jellyfish gene, a yeast gene, and so on.

A DNA synthesiser does not require enzymes or a template strand.

True. Instruments called DNA synthesisers can join nucleotide sub-units in a predefined order to produce DNA segments with lengths greater than 100 bases. The chemical synthesis of DNA does not require a template strand nor does it require the enzyme DNA polymerase.

CRISPR can be used to silence a gene.

True. One CRISPR technique is a process that can result in a random insertion or deletion of one or two bases, producing a frameshift mutation. A frameshift mutation can either disable a gene or can produce a STOP signal. So, the aim of this type of process is to disable or silence a gene.

The base sequence of a probe is complementary to part of the base sequence of its DNA target.

True. Probes typically consist of short single-stranded nucleic acids, either DNA or RNA, which can locate target DNA through complementary base pairing.

Target DNA must be denatured before it can be located with a probe.

True. Target DNA must be denatured (made single stranded) before it can be located with a probe. The probe is single stranded and it can pair with a complementary base sequence in the single-stranded target DNA.

In a long DNA sequence, it is reasonable to predict that there would be more cut sites for Hae III than for Hin dIII.

True. The number of nucleotide base pairs (bps) in a restriction site varies from four to about eight base pairs (bps). This length determines how frequently a restriction enzyme is likely to cut a random sequence of DNA, or in other words, the average distance between cuts. For example, restriction enzymes with a 4-bp recognition sequence, such as Hae III, are expected on average to cut about every 256 bps along a sequence. In contrast, a restriction enzyme with a 6-bp recognition sequence, such as Hin dIII, will on average cut about every 4096 bps.

GENE THERAPY

Using genetic engineering to replace an unhealthy gene with a healthy gene.

RESTRICTION ENZYME

What cuts DNA open at specific sites for recombination (like scissors).

For transformation to occur, the DNA taken up by bacteria must be ________ in their phenotypes.

expressed

The replication of plasmids is ________ of the replication of the main bacterial chromosome.

independent

Temporary joins can be made permanent through the use of ________.

ligase

A vector commonly used in transformation experiments is a ________.

plasmid

Sticky ends join temporarily through the formation of ________.

weak hydrogen bonds

List these steps in the appropriate order to make a recombinant plasmid

• identify relevant gene • construct recombinant DNA vector • transfer recombinant DNA into host cells • copy recombinant DNA • screen transformed cells.