Genetic Technologies

The polymerase chain reaction (PCR) is heated to about 95 °C in its first step. What is the purpose of this high temperature?

• ATo break the hydrogen bonds between the two DNA strands, separating them (denaturation)✓
• BTo activate the heat-stable DNA polymerase (Taq) enzyme
• CTo allow primers to bind to the single strands
• DTo join the new nucleotides together into a continuous strand (elongation)

Answer: Heating to ~95 °C breaks the hydrogen bonds holding the two strands of the DNA double helix together, separating (denaturing) it into single strands that can act as templates. Primer annealing occurs later at a lower temperature.

Why is a thermostable DNA polymerase, such as Taq polymerase, used in PCR?

• AIt is not denatured by the high (≈95 °C) temperatures used in each cycle✓
• BIt works fastest at low temperatures around 4 °C
• CIt can extend DNA fragments without needing primers
• DIt removes the primers once the final cycle is complete

Answer: Taq polymerase is isolated from a thermophilic bacterium and is heat-stable, so it survives the repeated 95 °C denaturation steps. A normal polymerase would be denatured and would need replacing each cycle.

Reverse transcriptase is used to make a gene from mRNA. The single-stranded molecule it produces directly from the mRNA template is called:

• Aa ribosomal RNA (rRNA) transcript
• Bcomplementary DNA (cDNA)✓
• Ca bacterial plasmid (a vector)
• Dmessenger RNA

Answer: Reverse transcriptase catalyses the synthesis of a single strand of complementary DNA (cDNA) using mRNA as the template. DNA polymerase can then make this double-stranded to give the gene.

What is one advantage of using mRNA and reverse transcriptase to obtain a human gene for insertion into a bacterium, rather than cutting the gene directly from genomic DNA?

• AThe cDNA produced contains introns that the bacterium needs
• BmRNA is more stable than DNA, which makes it easier to store
• CReverse transcriptase also inserts the finished gene directly into the bacterial chromosome, so a vector and ligase are not required
• DThe cDNA lacks introns, so it can be transcribed and translated correctly by the bacterium✓

Answer: mRNA from a mature transcript has already had its introns removed by splicing. cDNA made from it therefore contains only exons, so prokaryotes (which cannot splice out introns) can express the protein correctly.

Restriction endonucleases cut DNA at specific points. A particular enzyme cuts the two strands at slightly different positions, leaving short single-stranded overhangs. These overhangs are known as:

• Ablunt ends
• Bprimers
• Cpalindromes
• Dsticky ends✓

Answer: When a restriction enzyme makes a staggered cut within its recognition site, it produces sticky ends: short single-stranded overhangs. Complementary sticky ends on different DNA fragments can base-pair, aiding recombination.

What is the role of DNA ligase in producing recombinant DNA?

• AIt joins the sugar-phosphate backbones of the DNA fragments by forming phosphodiester bonds✓
• BIt cuts the DNA at specific palindromic recognition sequences (restriction sites), leaving complementary sticky ends
• CIt separates the two strands of the double helix by breaking the hydrogen bonds between complementary bases
• DIt copies the inserted gene many times over inside the host cell, producing multiple copies of the recombinant plasmid

Answer: After complementary sticky ends have base-paired, DNA ligase catalyses the formation of phosphodiester bonds in the sugar-phosphate backbone, sealing the gene into the vector to create a stable recombinant DNA molecule.

A gene (DNA) probe is used to locate a specific allele in a sample of DNA. Which feature of the probe allows it to identify the target sequence?

• AIt is a double-stranded molecule that has exactly the same base sequence as the whole of the chromosome being tested
• BIt is an enzyme that recognises the target sequence, cuts it, and releases a labelled fragment that can be seen on a gel
• CIt is a short single-stranded piece of DNA with a base sequence complementary to the target, carrying a label✓
• DIt is a monoclonal antibody raised against the target allele, which binds to the DNA and then shows up under ultraviolet light

Answer: A gene probe is a short single-stranded length of DNA with a base sequence complementary to the target sequence, and it carries a radioactive or fluorescent label. It binds (hybridises) to the target by complementary base pairing, revealing its position.

In the chain-termination (Sanger) method of DNA sequencing, modified nucleotides are added to the reaction. What is the effect of incorporating one of these modified (dideoxy) nucleotides into a growing DNA strand?

• AIt speeds up the addition of further nucleotides by DNA polymerase
• BIt cuts the DNA strand at that point (nicking)
• CIt causes the strand to fold into a double helix
• DIt stops further nucleotides being added, terminating that strand at a known base✓

Answer: Each modified (dideoxy) nucleotide lacks the 3′-OH group needed to form the next phosphodiester bond, so once incorporated it terminates strand synthesis. Each terminating base is labelled, producing fragments of different lengths that reveal the sequence.

Genetic fingerprinting compares DNA between individuals. Which regions of the genome are analysed because they vary greatly from person to person?

• AThe exons of protein-coding genes
• BRepeating, non-coding base sequences such as short tandem repeats (variable number tandem repeats)✓
• CThe promoter regions of housekeeping genes, which are transcribed continuously in the cells of the body
• DThe mitochondrial ribosomal RNA genes

Answer: Genetic fingerprinting targets non-coding regions containing variable numbers of tandem repeats (short tandem repeats). The number of repeats at each locus varies highly between individuals, so the overall pattern is effectively unique (except in identical twins).

Gene therapy aims to treat genetic disorders. In which situation would the therapeutic effect NOT be passed on to a patient's children?

• AWhen a functioning allele is introduced into the gametes or a very early embryo
• BWhen a functioning allele is introduced into the patient's somatic (body) cells✓
• CWhenever a viral vector is used
• DOnly when the disorder is caused by a dominant allele

Answer: Somatic gene therapy alters only body cells, so the change is not present in gametes and cannot be inherited. Germ-line therapy (altering gametes/embryos) would be heritable, but it is not permitted for humans for ethical reasons.

Cystic fibrosis is caused by two recessive alleles. Why might somatic-cell gene therapy that adds a normal allele be effective even though the faulty allele is still present?

• AThe added allele deletes the faulty allele from the chromosome, so a single working copy remains
• BThe added allele prevents the cell from dividing (mitotic arrest)
• CThe added allele repairs the faulty allele (gene editing), changing its base sequence
• DThe added normal (dominant) allele is expressed and produces a functioning protein, masking the recessive faulty allele✓

Answer: Adding a normal (dominant) allele lets the cell produce the functioning protein (e.g. the CFTR channel). Because the normal allele is dominant, its product masks the effect of the recessive faulty allele, relieving symptoms even though the faulty allele remains.

What does a restriction endonuclease do?

• ACuts DNA at a specific recognition sequence✓
• BJoins DNA fragments at a specific recognition sequence
• CCopies DNA from a specific recognition sequence
• DReads the base sequence of a DNA fragment

Answer: Restriction enzymes recognise short palindromic sequences and cut there, often leaving single-stranded sticky ends that make joining easier. Ligase does the joining and polymerase the copying.

Why is reverse transcriptase used when producing a gene from mRNA?

• AIt synthesises complementary DNA from an mRNA template✓
• BIt amplifies the mRNA so that many copies become available
• CIt removes the introns from the DNA before it is inserted
• DIt joins the new gene into the plasmid vector permanently

Answer: Reverse transcriptase makes cDNA from mRNA. Because mature mRNA has already had its introns spliced out, the resulting gene can be expressed in a prokaryote, which cannot splice. Amplification is done by PCR and joining by ligase.

Why is reverse transcriptase useful in making a gene for insertion?

• AIt builds mRNA from a DNA template, avoiding introns
• BIt builds DNA from an mRNA template, avoiding introns✓
• CIt removes introns from a length of genomic DNA directly
• DIt amplifies the number of copies of the required gene

Answer: Mature mRNA has already had its introns spliced out, so copying it back into cDNA gives a gene a bacterium can express — bacteria cannot splice. Amplification is PCR's job.

What is the purpose of the polymerase chain reaction?

• ASeparating DNA fragments according to their length
• BCutting DNA at specific recognition sequences
• CAmplifying a DNA sample into many identical copies✓
• DDetermining the base sequence of a DNA fragment

Answer: PCR cycles through denaturing at about 95 °C, annealing primers at around 55 °C and extending with a heat-stable polymerase, roughly doubling the DNA each cycle. It is why a trace sample can be analysed at all.

Why is Taq polymerase used in PCR rather than human DNA polymerase?

• AIt is not denatured by the high temperatures used✓
• BIt works far faster than any other known polymerase
• CIt can build DNA without needing any primer at all
• DIt copies RNA into DNA as well as copying DNA

Answer: PCR heats to about 95 °C every cycle, which would denature most enzymes. Taq comes from a thermophilic bacterium and survives, so it need not be replaced each round — the single change that made PCR practical.

How does gel electrophoresis separate DNA fragments?

• ALarger fragments travel further through the gel
• BFragments separate according to their base sequence
• CSmaller fragments travel further through the gel✓
• DFragments separate according to their overall charge

Answer: DNA is negatively charged and moves towards the anode; the gel resists larger fragments more, so small ones travel furthest in a given time. All DNA carries a similar charge per unit length, so charge does not do the separating.

What is a marker gene used for?

• AIdentifying where the plasmid should be cut open
• BIncreasing the rate at which the plasmid replicates
• CIdentifying which cells have taken up the plasmid✓
• DPreventing the plasmid being expelled from the cell

Answer: A marker such as antibiotic resistance or fluorescence lets you select only the transformed cells, since uptake is inefficient and most cells take up nothing. Without it you could not tell which colonies matter.

What is a DNA probe used to detect?

• AA specific protein produced by a gene
• BThe total quantity of DNA in a sample
• CA specific complementary base sequence✓
• DThe rate at which a gene is transcribed

Answer: A probe is a short labelled single strand that hybridises only with its complementary sequence, revealing whether a particular allele is present. It detects DNA sequence, not protein or transcription rate.

What is genetic fingerprinting based on?

• AVariable numbers of repeated protein-coding sequences
• BThe complete base sequence of an individual's genome
• CDifferences in the number of chromosomes between people
• DVariable numbers of repeated non-coding sequences✓

Answer: Variable number tandem repeats in non-coding DNA differ greatly between individuals, so the banding pattern is effectively unique except in identical twins. Coding regions are far too conserved to distinguish people.