GATE Life Sciences Chemistry of Biomolecules Syllabus

What is Chargaff's rule for DNA base composition?

In double-stranded DNA, $[\text{A}] = [\text{T}]$ and $[\text{G}] = [\text{C}]$, therefore purines = pyrimidines: $[\text{A}] + [\text{G}] = [\text{C}] + [\text{T}]$.

In which direction is a new DNA strand synthesized, and what is read as the template?

DNA polymerase synthesizes the new strand in the $5' \to 3'$ direction, reading the template strand in the $3' \to 5'$ direction.

What type of bond joins nucleotides along a DNA strand?

A 3',5'-phosphodiester bond linking the 3'-OH of one sugar to the 5'-phosphate of the next nucleotide.

Name the two classic DNA sequencing methods and their inventors.

The Maxam–Gilbert method (chemical degradation) and the Sanger method (chain-termination / dideoxy, enzymatic).

On what principle does the Maxam–Gilbert (chemical) DNA sequencing method rely?

It relies on base-specific chemical modification followed by cleavage of the phosphodiester backbone at the modified bases, generating labeled fragments of different lengths that are separated by gel electrophoresis.

Which chemicals are used in Maxam–Gilbert sequencing to modify specific bases?

Dimethyl sulfate (DMS) modifies G (and A under acid); formic acid attacks purines (A+G); hydrazine attacks pyrimidines (C+T), and hydrazine with high salt (NaCl) targets C only. Piperidine then cleaves the backbone at modified sites.

What is the key reagent that causes chain termination in Sanger (dideoxy) sequencing?

2',3'-dideoxynucleoside triphosphates (ddNTPs), which lack the 3'-OH group required to form the next phosphodiester bond, so chain elongation stops once a ddNTP is incorporated.

Why does the absence of a 3'-OH in a ddNTP terminate DNA synthesis?

Chain elongation requires a free 3'-OH on the growing strand to attack the incoming nucleotide's $\alpha$-phosphate. A ddNTP has $\ce{H}$ instead of $\ce{OH}$ at the 3' position, so no further phosphodiester bond can form and the chain terminates.

Compare Maxam–Gilbert and Sanger methods: which is enzymatic and which is chemical?

Maxam–Gilbert is a chemical method (no DNA polymerase; uses base-specific cleavage). Sanger is an enzymatic method (uses DNA polymerase, a primer, dNTPs, and chain-terminating ddNTPs).

What components are required in a classic Sanger sequencing reaction?

A single-stranded DNA template, a short primer (with free 3'-OH), DNA polymerase, the four dNTPs, and a small amount of one chain-terminating ddNTP (four separate reactions, one per ddNTP, in the original method).

How are DNA fragments resolved and read in classic gel-based sequencing?

Fragments are separated by high-resolution denaturing polyacrylamide gel electrophoresis according to length (single-nucleotide resolution); the sequence is read from the bottom (shortest, 5') to the top (longest).

What is a hexose? Give two important examples.

A hexose is a monosaccharide containing six carbon atoms (molecular formula $\ce{C6H12O6}$). Examples: glucose and fructose (also galactose, mannose).

Distinguish between an aldohexose and a ketohexose with examples.

An aldohexose has an aldehyde group ($\ce{-CHO}$) at C-1 (e.g. glucose, galactose); a ketohexose has a ketone group ($\ce{C=O}$) at C-2 (e.g. fructose).

What is the difference between the $\alpha$ and $\beta$ anomers of glucose?

They differ in configuration at the anomeric carbon (C-1). In $\alpha$-D-glucose the C-1 $\ce{-OH}$ is on the opposite side (trans, down) to the C-6 reference; in $\beta$-D-glucose it is on the same side (cis, up) in the Haworth projection.

What is mutarotation in the context of hexoses?

Mutarotation is the spontaneous change in optical rotation that occurs when one anomer ($\alpha$ or $\beta$) of a sugar interconverts to an equilibrium mixture of both anomers through the open-chain form in solution.

Define epimers and give a hexose example.

Epimers are diastereomers that differ in configuration at a single chiral carbon. For example, D-glucose and D-galactose are C-4 epimers; D-glucose and D-mannose are C-2 epimers.

What makes a sugar a 'reducing sugar', and which test detects it?

A reducing sugar has a free anomeric carbon (free aldehyde or ketone group capable of being oxidized). It gives a positive Benedict's, Fehling's, or Tollens' test; e.g. glucose and fructose are reducing sugars.

How many stereoisomers are possible for an aldohexose, and why?

An aldohexose has 4 chiral centers (C-2, C-3, C-4, C-5), so it has $2^{4} = 16$ stereoisomers (8 D-forms and 8 L-forms).

What is a triglyceride (triacylglycerol) chemically?

A triglyceride is an ester of glycerol (a trihydric alcohol) with three fatty acid molecules, formed by esterification of the three hydroxyl groups; it is the main storage form of fat.

Write the general reaction of triglyceride formation.

$$\ce{Glycerol + 3\,Fatty\ acids -> Triglyceride + 3\,H2O}$$ Each fatty acid carboxyl condenses with a glycerol hydroxyl to form an ester bond, releasing water.

What is saponification of a triglyceride?

Saponification is the alkaline (e.g. $\ce{NaOH}$/$\ce{KOH}$) hydrolysis of a triglyceride yielding glycerol and the salts of the fatty acids (soaps).

Define the saponification number of a fat or oil.

The saponification number is the number of milligrams of $\ce{KOH}$ required to saponify 1 gram of fat or oil; it is inversely related to the average molecular weight (chain length) of the fatty acids.

Define the iodine number (iodine value) of a fat.

The iodine number is the number of grams of iodine ($\ce{I2}$) that can be added to 100 grams of fat; it measures the degree of unsaturation (number of C=C double bonds) — higher value means more unsaturation.

Why do unsaturated triglycerides (oils) have lower melting points than saturated ones (fats)?

Cis double bonds introduce kinks that prevent tight packing of the fatty acid chains, weakening van der Waals interactions and lowering the melting point, so unsaturated triglycerides are liquid (oils) at room temperature while saturated ones are solid (fats).

On what property does ion-exchange chromatography separate molecules?

It separates molecules based on their net surface charge — by reversible electrostatic interaction between charged groups on the analyte and oppositely charged groups fixed on the stationary phase (resin).

Distinguish between a cation exchanger and an anion exchanger.

A cation exchanger carries negatively charged groups (e.g. $\ce{-SO3^-}$, carboxymethyl/CM) and binds positively charged (cationic) molecules. An anion exchanger carries positively charged groups (e.g. $\ce{-N(CH3)3^+}$, DEAE) and binds negatively charged (anionic) molecules.

How are bound molecules typically eluted in ion-exchange chromatography?

By increasing the salt concentration (ionic strength) of the buffer, which competes for the charged binding sites, or by changing the pH to alter the net charge of the bound molecules — usually applied as a gradient.

On what basis does gel filtration (size-exclusion) chromatography separate molecules?

It separates molecules according to their size (and shape / hydrodynamic radius / molecular weight), not charge — using a porous gel matrix that excludes large molecules and retains small ones.

In gel filtration, which molecules elute first and why?

Large molecules elute first because they are too big to enter the pores of the beads and pass through the void volume (the spaces between beads), traveling a shorter path; small molecules enter the pores, take a longer path, and elute later.

What do the void volume ($V_0$) and total volume ($V_t$) represent in gel filtration?

$V_0$ (void volume) is the volume of solvent between the gel beads, at which fully excluded (largest) molecules elute. $V_t$ is the total column volume; molecules small enough to fully penetrate all pores elute near $V_t$. The elution volume $V_e$ of a molecule depends on its size, with $V_0 < V_e < V_t$.

Name common matrix materials used in gel filtration chromatography.

Cross-linked dextran (Sephadex), agarose (Sepharose), and polyacrylamide (Bio-Gel P) beads of defined pore size.

State the Beer–Lambert law in equation form and define each term.

$$A = \varepsilon \, c \, l$$ where $A$ is absorbance, $\varepsilon$ is the molar absorptivity (extinction coefficient, $\text{M}^{-1}\text{cm}^{-1}$), $c$ is the molar concentration, and $l$ is the path length of the cuvette (cm).

How is absorbance ($A$) related to transmittance ($T$) and to incident/transmitted light intensity?

$$A = -\log_{10}(T) = \log_{10}\!\left(\frac{I_0}{I}\right)$$ where $I_0$ is the incident light intensity and $I$ is the transmitted intensity; transmittance $T = \frac{I}{I_0}$.

According to the Beer–Lambert law, what is the relationship between absorbance and concentration, and why is it useful?

Absorbance is directly proportional to concentration ($A \propto c$) at fixed path length and wavelength. This linear relationship allows the unknown concentration of a colored or UV-absorbing solute to be determined spectrophotometrically from a calibration (standard) curve.

Under what conditions does the Beer–Lambert law deviate from linearity?

At high concentrations (solute–solute interactions, refractive index changes), with non-monochromatic light, due to scattering by turbid/particulate samples, chemical changes (association/dissociation, fluorescence), or stray light — causing the $A$ vs $c$ plot to deviate from a straight line.

At what wavelength do proteins typically absorb in the UV, and which residues are responsible?

Proteins absorb maximally around $280\ \text{nm}$ due to the aromatic side chains of tryptophan and tyrosine (and weakly phenylalanine); the peptide bond itself absorbs around $190$–$220\ \text{nm}$.

At what wavelength do nucleic acids (DNA/RNA) absorb maximally, and why?

Nucleic acids absorb maximally at $260\ \text{nm}$ due to the aromatic purine and pyrimidine bases; the $A_{260}/A_{280}$ ratio is used to assess DNA/protein purity (pure DNA $\approx 1.8$).

What is the role of piperidine in Maxam–Gilbert sequencing?

After base-specific chemical modification, hot piperidine catalyzes cleavage of the DNA backbone at the modified (and thereby displaced) bases, producing the labeled fragments that are then size-separated.

Why is one peptide-bond plane considered rigid and planar?

The peptide ($\ce{C-N}$) bond has partial double-bond character due to resonance delocalization of the carbonyl/amide electrons, restricting rotation and keeping the six atoms of the peptide group ($\ce{C_\alpha, C, O, N, H, C_\alpha}$) coplanar; the trans configuration is generally favored.