Molecular Biology Techniques Questions and Answers

Answer: C

Recruitment of epigenetic factors on DNA is detected by chromatin immunoprecipitation followed by PCR (ChIP-PCR).

In ChIP, proteins are cross-linked to the DNA they are bound to in living cells, usually with formaldehyde. The chromatin is sheared, and an antibody against the factor of interest, such as a histone-modifying enzyme or a modified histone, pulls down the protein with its attached DNA. After reversing the cross-links, PCR (or sequencing, in ChIP-seq) shows which DNA regions the factor occupied.

Plain immunoprecipitation and immunoblotting detect the protein but not where on the DNA it sits. Immunofluorescence shows where the protein is in the nucleus, not which sequences it binds.

Answer: D

Heparin columns are used to purify DNA polymerases.

Heparin is a strongly negatively charged sulphated polysaccharide that resembles the phosphate backbone of nucleic acids. Proteins that bind DNA, such as DNA polymerases, restriction enzymes and many transcription factors, therefore bind heparin and can be eluted with a salt gradient. Heparin also binds coagulation factors and lipoprotein lipases.

The other targets use different ligands. NAD⁺-dependent dehydrogenases are purified on dye columns such as Cibacron Blue or on 5'-AMP agarose. Glycoproteins are captured by lectins such as concanavalin A, and eukaryotic mRNA by oligo(dT) cellulose, which binds the poly(A) tail.

Answer: A

The technique is the zoo blot.

A zoo blot is a Southern blot carrying DNA from several different animal species, probed with a cloned gene or DNA fragment from one species. Hybridisation in many species shows that the sequence is conserved, which suggests it is a protein-coding gene; it was used, for example, in identifying the gene for Duchenne muscular dystrophy.

'Garden blot', 'phylogenetic blot' and 'animal profiling' are not names of a blotting technique; they play on the idea of comparing species.

Answer: A

Southern blotting depends on similarities between the sequences of probe DNA and experimental DNA.

In Southern blotting (E. M. Southern, 1975), DNA fragments are separated by gel electrophoresis, denatured, transferred to a membrane and exposed to a labelled single-stranded probe. The probe base-pairs only with fragments whose sequence is complementary to it, so the method detects a particular sequence by hybridisation.

Molecular mass and amino acid sequence of proteins are the basis of Western blotting, not Southern. Dissimilarity between RNA and DNA is not what the method detects.

Answer: B

SDS is used because it binds to proteins and confers a uniform negative charge density, so they move in the gel according to size.

Sodium dodecyl sulphate is an anionic detergent. It unfolds polypeptides and coats them at a roughly constant ratio of about 1.4 g of SDS per gram of protein. The protein's own charge is swamped, every chain carries a similar charge per unit length, and migration through the polyacrylamide sieve then depends almost only on molecular mass.

SDS does solubilise proteins, but that alone would not make separation by mass possible, so the first option misses the point. It is not used to change the buffer's surface tension, and far from stabilising proteins it denatures them.

Answer: C

The best technique is autoradiography.

In autoradiography the specimen, a tissue section, gel or blot, is placed against photographic emulsion or film. Radiation from the isotope exposes the emulsion just where the labelled molecules are, giving an image of their precise location. It is how pulse-chase experiments traced newly made proteins through the ER and Golgi.

Ultracentrifugation separates particles by size and density but does not show location in a specimen. Atomic force microscopy maps surface topography, and fluorescence microscopy detects fluorophores, not radioactive decay.

Answer: D

The sedimentation coefficient increases with a decrease in partial specific volume.

The Svedberg equation gives the sedimentation coefficient as:

\[ s = \frac{M(1 - \bar{v}\rho)}{N_A f} \]

where \(M\) is molar mass, \(\bar{v}\) the partial specific volume, \(\rho\) the solvent density and \(f\) the frictional coefficient. A smaller \(\bar{v}\) (a denser particle) makes the buoyancy term \(1 - \bar{v}\rho\) larger, so \(s\) rises.

The other changes all lower \(s\): a denser solvent increases buoyancy, more friction slows the particle, and less mass reduces the driving force.

Answer: A

There will be 4 labelled and 6 unlabelled fragments.

Three EcoRI sites divide the molecule into 4 segments. 'Possible fragments' means every fragment a partial digest can give, any run of adjacent segments. With \(n\) segments the count is:

\[ \frac{n(n+1)}{2} = \frac{4 \times 5}{2} = 10 \]

A fragment is labelled only if it includes the labelled end. Those are the runs starting at that end: segment 1, segments 1-2, 1-3 and 1-4, so 4 are labelled and \(10 - 4 = 6\) are not.

A complete digest would give only 4 fragments, 1 labelled. Options with 6 labelled reverse the count, and 3 and 3 or 3 and 5 have no consistent total.

Answer: C

RAPD is a dominant marker.

Random amplified polymorphic DNA uses single short primers of arbitrary sequence in PCR. A polymorphism shows as the presence or absence of a band: if the primer site is lost in one allele, that allele gives no product. A heterozygote has one copy of the band-forming allele and shows the same band as the homozygote, so the two cannot be told apart. That presence/absence behaviour is what is meant by a dominant marker.

Co-dominant markers, such as RFLPs and microsatellites (SSRs), show both alleles as separate bands and distinguish heterozygotes. 'Recessive' and 'partially dominant' are not used to describe RAPD.

Answer: A

The protein binding site is identified by DNA footprinting.

In footprinting, a DNA fragment labelled at one end is lightly digested with DNase I (or cut with hydroxyl radicals) with and without the bound protein. The protein shields the bases it covers, so on a sequencing gel the ladder of fragments shows a gap, the 'footprint', exactly at the binding site, which can be read against a sequence ladder.

A mobility shift assay (EMSA) shows that a protein binds a DNA fragment, by slowing its migration, but not where on the fragment. Western blotting detects proteins with antibodies, and DNA fingerprinting identifies individuals from repeat polymorphisms.