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 DNA polymerase enzyme
• CTo allow primers to bind to the single strands
• DTo join the new nucleotides together

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 join DNA fragments without primers
• DIt removes primers at the end of the reaction

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:

• Aribosomal RNA
• Bcomplementary DNA (cDNA)✓
• Ca plasmid
• 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 and easier to store than DNA
• CReverse transcriptase also inserts the gene into the bacterial chromosome
• 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 recognition sequences
• CIt separates the two strands of the DNA
• DIt copies the inserted gene many times

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 identical to the whole chromosome
• BIt is an enzyme that cuts the target sequence
• CIt is a short single-stranded piece of DNA with a base sequence complementary to the target, carrying a label✓
• DIt is an antibody specific to the DNA

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
• BIt cuts the DNA at that point
• 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
• 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
• BThe added allele prevents the cell from dividing
• CThe added allele changes the faulty allele into a normal one
• 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.