MOLBIO COMPILED PRELIMS REVIEWER

MOLBIO COMPILED PRELIMS REVIEWER

MLS 420 — Molecular Biology: Nucleic Acids, DNA Mutation & Nucleic Acid Isolation

PART 1: NUCLEIC ACIDS

Key Scientists & Contributions

DNA vs. RNA — Quick Comparison

Parts of a Nucleotide

3 components:

1. Pentose Sugar — Deoxyribose (DNA) or Ribose (RNA)

2. Phosphate Group — gives acidic property; forms the backbone (negatively charged)

3. Nitrogenous Base — gives identity

Formation steps:

• Base + Sugar → Nucleoside (Glycosidic bond)

• Nucleoside + Phosphate → Nucleotide (Phosphoester bond)

• Nucleotide + Nucleotide → Nucleic Acid Chain (Phosphodiester bond)

Types of Bonds

Strength:

• Strongest → Phosphodiester bond (backbone; outer protection)

• Weakest → Hydrogen bond (inside; broken during replication/denaturation)

Hydrogen bond count:

• A = T → 2 hydrogen bonds

• G ≡ C → 3 hydrogen bonds

Nitrogenous Bases

Purines (double ring) — PuGA:

• Adenine — found in DNA and RNA

• Guanine — found in DNA and RNA

Pyrimidines (single ring) — CUTiePy:

• Cytosine — found in DNA and RNA

• Thymine — found in DNA only

• Uracil — found in RNA only

DNA Structure

Polarity: Anti-parallel (one strand 5'→3', the other 3'→5')

Grooves:

• Major groove — large curve; nucleotides far apart; what Rosalind Franklin observed

• Minor groove — small curve; nucleotides close together

Types of DNA:

Formation of Chromosomes (6 Steps)

1. DNA double helix — 2 nm (naked DNA)

2. Nucleosome — DNA wraps around histones ("beads-on-a-string") — 11 nm

3. Solenoid/Chromatin — nucleosomes compact; linked by condensin — 30 nm

4. Supercoiled chromatin fibers — several condensin units — 300 nm

5. Sister chromatid — densely condensed — 700 nm

6. Duplicated (mitotic) chromosome — two sister chromatids linked by cohesin — 1400 nm

PART 2: RNA TYPES

Protein Coding:

A. mRNA (Messenger RNA)

• Most heterogeneous RNA — 5% of total RNA

• Carries genetic info from DNA to ribosomes

• Template for protein synthesis

• In eukaryotes: has methylguanosine cap (5' end) + poly-A tail (3' end) — protection from nucleases

• Undergoes splicing (removes introns/bad codes, retains exons/good codes) — unique to eukaryotes

Non-Protein Coding:

B. rRNA (Ribosomal RNA)

• Most abundant — 80% of total RNA ("r = rami-raming!")

• Forms and functions in ribosomes

• Two subunits:

  • Small subunit — positions/aligns mRNA (usher); proofreads codon-anticodon matching

    • Prokaryotes: 30S (16S) | Eukaryotes: 40S (18S)

  • Large subunit — forms peptide bonds (catalyst)

    • Prokaryotes: 50S (23S) | Eukaryotes: 60S (28S)

• Target of antibiotics (gentamicin, erythromycin, tetracycline)

C. tRNA (Transfer RNA)

• Smallest RNA — 15% of total RNA ("t = tiny")

• Adapter/translator molecule — speaks both nucleotide and amino acid languages

• Contains anticodon (3'→5' direction)

• Cloverleaf shape in 2D

• ALL tRNA end with CCA (attachment point for amino acids)

• At least 20 different species

D. snRNA (Small Nuclear RNA)

• Functions in mRNA and rRNA processing

• Splices exons together to form mature mRNA

• Removes introns ("bad codes")

E. miRNA (Micro RNA)

• Interacts with 3' untranslated region of mRNA

• Induces mRNA degradation and translational repression

• Regulates/prevents overproduction of proteins

F. siRNA (Small Interfering RNA)

• Double-stranded RNA (20–24 bp) — "short interfering RNA"

• Interferes with gene expression

• Induces mRNA degradation

• Enzyme Dicer converts dsRNA/hairpin RNA into siRNA

G. lncRNA (Long Non-Coding RNA)

• Non-coding transcripts >200 nucleotides

• Involved in cell differentiation, development

• Maintains telomere length (TERC and TERRA)

• Longer telomere = longer life

PART 3: PROTEINS

Amino Acid Chain Names

Smallest protein: Oxytocin (9 AAs) | Largest protein: Titin (25,000 AAs)

Structural Organization of Proteins

Key notes:

• Mutations and substitutions occur at the primary structure

• Example: Sickle Cell Anemia — glutamine substituted by valine at 6th position

• Molecular chaperones assist folding but do NOT determine final structure

Central Dogma

DNA → (Transcription) → RNA → (Translation) → Protein

• Replication — DNA → DNA (nucleus)

• Transcription — DNA → mRNA (nucleus)

• Translation — mRNA → Protein (ribosome/cytoplasm)

• Reverse Transcription — RNA → DNA (retroviruses)

Genomics vs. Proteomics

PART 4: DNA MUTATION

Transcription & Translation Basics

• Sense strand — 5' to 3'; same sequence as mRNA

• Antisense strand (template) — 3' to 5'; mRNA is based on this strand

• mRNA — always 5' to 3'; contains codons

• tRNA — contains anticodons (3' to 5')

• New strands always synthesized in the 5' to 3' direction (due to hydroxyl group at 3')

• Each codon = 3 nucleotides = codes for 1 amino acid

• Total codons: 64 | Recognized by tRNA: 61 | Stop codons: 3 (UAA, UAG, UGA)

• Start codon: AUG (Methionine)

Wobble Hypothesis (Francis Crick)

• First two codon positions — always follow complementary base pairing

• Third position — can "wobble" (non-Watson-Crick pairing)

• This is why only 20 amino acids exist despite 64 codons

• Silent mutations usually occur at the 3rd (wobble) position

Sources of Mutation

Spontaneous Mutation (natural):

1. Tautomeric shift — base changes chemical structure → mispairing

2. Depurination — 10,000 purines lost per cell cycle; glycosidic bond interrupted

3. Deamination — amino group removed from cytosine → becomes uracil

4. Oxidative stress — toxic byproducts (superoxide radicals, H₂O₂) damage bases

Induced Mutation (external/mutagenesis):

1. Base replacement (analogs) — chemical mimics a base; e.g., bromouracil mimics guanine

2. Base damage — physical distortion or broken linkage; often caused by UV radiation

3. Base alteration — bases modified by alkylating/hydroxylating agents; e.g., EMS adds ethyl group to guanine

Types of Mutation

A. Gene Mutation

Point Mutation (Substitution):

Functional types of Point Mutation:

Frameshift Mutation (Indels):

• Insertion — addition of a base; shifts reading frame

• Deletion — removal of a base; shifts reading frame

• If 3 bases (1 codon) inserted/deleted → NO frameshift, only AA added/removed

• Example: Tay-Sachs disease — deletion of cytosine in HEXA gene

B. Chromosome Mutation

• Paracentric inversion — without centromere

• Pericentric inversion — with centromere

• Terminal deletion — end of chromosome deleted

• Interstitial deletion — within the chromosome deleted

C. Genome Mutation

Euploid = normal chromosome number (46 chromosomes / 23 pairs)

Aneuploidy (change in single chromosome number):

• Monosomy — 2n − 1 (one chromosome missing its pair)

• Trisomy — 2n + 1 (extra chromosome); example: Down syndrome

Polyploidy (abnormal number of entire chromosome sets):

• Normal humans = diploid (2n)

• More than diploid in humans = pathologic

• Plants are classic examples of polyploidy (explains their ability to regrow)

D. Other Mutations

• Trinucleotide repeat expansion — overproduction of same amino acid

• Extensive insertions/deletions — major form of indel

• Major chromosomal rearrangements — two or more chromosome mutations simultaneously

PART 5: NUCLEIC ACID ISOLATION

Workflow in Molecular Biology

Sample Collection → Nucleic Acid Extraction → Quality Check (Gel electrophoresis) + Quantification (Nanodrop) → PCR Amplification → Gel Electrophoresis → Downstream Applications

Goals of Nucleic Acid Isolation

• Purity — DNA/RNA only; no contaminants

• Quality — intact, not degraded

• Quantity — sufficient for downstream applications

General Steps for Nucleic Acid Isolation

1. Cell Lysis / Tissue Homogenization

Factors affecting disruption strategy:

• Stability of molecules, size of sample, cohesion of cells, cell membrane type, presence of inhibitors (e.g., heme in RBCs inhibits PCR → use plasma, not whole blood)

2. Separation

• Chemical: Phenol (denatures proteins) + Chloroform (increases organic layer density)

• After centrifugation → 3 layers:

  • Aqueous phase — RNA (and sometimes DNA)

  • Interphase — DNA

  • Organic phase — proteins, lipids (contaminants)

• Take aqueous phase → proceed to precipitation

Precipitation:

• Add salt (monovalent cations: Na, K, NH₄ acetate, LiCl) → neutralizes negative phosphate backbone → promotes aggregation

• Add alcohol (95% EtOH or isopropanol) → precipitates nucleic acid (NA is NOT alcohol-soluble)

• Centrifuge → obtain pellet

3. Purification (Washing + Drying)

• Wash pellet with 70–80% ethanol (removes excess salt; water content dissolves salt)

• Wash at least twice

• Dry pellet (air dry or vacuum dry) — removes excess alcohol

• Do NOT over-dry (DNA breaks without moisture)

• Resuspend in:

  • DNA → TE buffer (Tris-EDTA)

  • RNA → Nuclease-free water

4. Concentrate (Optional)

• Done when sample is very small/light

• Before cell lysis → concentrates both DNA and RNA

• After cell lysis → concentrates either DNA or RNA

Nucleic Acid Isolation Methods

Chemical-Based (Traditional):

Solid-Phase Methods (Next Generation):

Steps: Lysis → Binding → Washing → Eluting

Nucleic Acid Stabilization

• Resuspend DNA in TE buffer | RNA in nuclease-free water

• Store at 5°C or −70°C

• AVOID −20°C (causes freeze-thaw cycles that degrade NA)

• Aliquot samples

• Use nuclease-free reagents and plasticware

Post-Isolation Treatments

• If DNA isolated → add RNase to remove RNA contamination

• If RNA isolated → add DNase to remove DNA contamination

• Check quality: Gel Electrophoresis

• Check quantity/purity: Nanodrop method

HIGH-YIELD MNEMONICS

COMMON CLINICAL EXAMPLES

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