Molecular Biology Flashcards

Flashcards for Molecular Biology review

What is Bioinformatics?

An interdisciplinary science combining biology, computer science, and mathematics to analyze and interpret biological data, particularly sequence data like DNA, RNA, and proteins, to extract meaningful insights from complex, large-scale datasets.

What are the three components of INSDC (International collaboration of sequence databases)?

DDBJ (Japan), GenBank (NCBI, USA), EMBL-EBI (Europe)

Name three major sequence browsers.

NCBI Genome Database, Ensembl Genome Browser, UCSC Genome Browser

What are some common sequence formats?

FASTA, GenBank, ENSEMBL

What tools does NCBI offer?

BLAST tool, PubMed, GenBank, and protein/gene databases

How is ENSEMBL searchable?

By gene symbol, chromosome coordinates, or phenotypic data

What do UCSC Browser tracks include?

Genomic annotations, comparative genomics, and sequence searches by position or gene symbol

What are homologues?

Genes sharing a common ancestry

What are orthologues?

Found in different species, usually with similar function

What are paralogues?

Duplicated within the same genome, may evolve new functions

What are mismatches and gaps in sequence alignments?

Mismatches = different nucleotides/amino acids; Gaps = insertions/deletions

Differentiate conservative vs radical substitutions in protein alignments

Conservative substitutions have similar side-chain properties, while radical substitutions have different properties.

What information does BLAST help determine?

Organism origin of a sequence, gene identity and mutations, and potential disease associations

What are the different type of BLAST searches?

blastn, blastp, blastx

What are the three steps of Standard PCR?

Denaturation, Annealing, Extension

What does RT-PCR do?

Converts RNA into cDNA before amplification

What does qPCR do?

Real-time measurement using fluorescent dyes or probes—used to quantify gene expression

What does Multiplex PCR do?

Uses multiple primer sets to amplify different targets in a single reaction

What does Nested PCR do?

Two rounds of PCR to improve specificity

What does Touchdown PCR do?

Improves specificity by gradually lowering annealing temperature

What is optimal primer length?

Typically 18–25 nucleotides; longer primers increase specificity

What is optimal melting temperature (Tm) for primers?

Should be similar for both primers (ideally 55–65°C)

What is the optimal GC content for primers?

Affects primer stability; 40–60% is optimal

What should be avoided in primer design?

Hairpin loops, primer dimers, and complementarity at 3’ ends (can cause mis-priming)

What are dNTPs?

Building blocks of DNA; optimal concentration is crucial to prevent errors

What is the role of Mg²⁺ ions in PCR?

Cofactor for DNA polymerase; too little = low yield, too much = non-specific products

What is the role of DNA polymerase in PCR?

Taq is standard, but high-fidelity enzymes (e.g., Pfu) offer higher accuracy

How does template quality impact PCR?

Impurities (e.g., phenol, ethanol) can inhibit PCR

What is NCBI Primer-BLAST?

Designs primers and checks specificity against entire genomes

How is BLASTn used in PCR assay development?

Ensures target region is unique—no off-target binding

What do tools like OligoCalc do?

Calculate Tm, GC%, and primer self-complementarity

When is RT-qPCR commonly used?

Gene expression studies and diagnostic testing (e.g. viral load quantification)

When does Multiplex PCR excel?

Pathogen panels and forensic identification

When is Touchdown PCR preferred?

Difficult templates and low-abundance targets

Name two methods of Real-time quantification in RT-qPCR.

SYBR Green dye and TaqMan probes

How do microarrays work?

thousands of DNA probes are immobilized on a chip, fluorescently labeled cDNA is hybridized to these probes, relative fluorescence intensity reflects expression level.

What does RNA-Seq offer?

Whole transcriptome coverage, including non-coding RNAs and splice variants.

What steps does raw reads undergo in RNA-Seq?

Quality control (FastQC), Alignment (HISAT2, STAR), Quantification (FeatureCounts, HTSeq), Differential expression analysis (DESeq2, edgeR)

What outputs does RNA-Seq provide?

counts per gene, FPKM, or TPM values

What practical skills should you learn for hands-on lab work?

sterile technique, pipette calibration, and reagent handling

What does BLASTn do?

Compares nucleotide query against nucleotide databases

What does BLASTp do?

Protein vs. protein database

What does blastx do?

Translates a nucleotide query into protein before comparison

Why clone genes?

To study gene function and expression, produce recombinant proteins, and modify genomes

What do restriction enzymes do?

Recognize specific short DNA sequences (usually palindromes) and cut the DNA at or near these sites

What are sticky ends?

Staggered cuts with overhangs—better for directional cloning

What are blunt ends?

Straight cuts with no overhangs—less efficient but more flexible

What is a Multiple Cloning Site (MCS)?

A short sequence in plasmids containing several restriction sites offering versatility in inserting DNA fragments using different enzymes

What is the function of the Origin of replication (Ori) in cloning vectors?

allows replication inside the host

What is the purpose of a selectable marker in cloning vectors?

usually an antibiotic resistance gene (e.g., amp^R)

What is the purpose of a reporter gene in cloning vectors?

e.g., lacZ for blue/white screening

What is the purpose of the MCS region in cloning vectors?

for insertion of the target gene

How does restriction digestion work for inserting DNA fragments into vectors?

using the same or compatible enzymes to produce matching ends.

How does ligation work for inserting DNA fragments into vectors?

DNA ligase joins the insert and vector by forming phosphodiester bonds, requiring ATP or NAD+ as a cofactor in the reaction

How does T/A cloning work for inserting DNA fragments into vectors?

PCR products made with Taq polymerase often have single “A” overhangs are inserted into "T” overhang vectors without restriction digestion.

What is transformation?

introducing recombinant DNA into bacteria (commonly E. coli)

How does the heat shock method work in transformation?

Cells briefly exposed to 42°C to encourage DNA uptake

How does electroporation work in transformation?

Electric field opens pores in bacterial membrane

How does antibiotic selection work?

Plating on antibiotic-containing agar ensures only transformed cells survive.

How does blue-white screening work?

Insert disrupts lacZ → colonies turn white (successful clone).; No insert → functional lacZ → blue colonies with X-gal.

What is the purpose of a negative control in gene cloning experiments?

Plasmid-only (no insert) to assess background ligation

What is the purpose of a positive control in gene cloning experiments?

A known successful insert to validate the system

What is the purpose of a no-enzyme control in gene cloning experiments?

Detects contamination or background resistance

What is the key enzyme in ligation?

T4 DNA ligase catalyzes phosphodiester bonds between vector and insert.

What kind of controls should you assess during ligation?

Insert-only should show no growth - verifies that insert alone doesn't confer resistance, Uncut vector should yield only blue colonies (non-recombinant).

How does chemical transformation using a heat shock work?

Chill cells + plasmid DNA on ice to allow binding, heat shock at 42°C for 30–60 seconds creates temporary pores, cells recover in SOC or LB broth to allow expression of antibiotic resistance gene.

How does electroporation work for transformation?

Delivers electrical pulses (~1.8 kV) to open membrane; must use salt-free buffer to avoid arcing.

How does Colony PCR work to screen colonies for correct cloning?

Pick individual colonies, use a small portion directly as PCR template; primers flank MCS region, presence/absence of insert alters expected product size.

How is Restriction digest of miniprep DNA used to screen colonies for correct cloning?

Digest plasmid from cultured colony using the same enzymes used during cloning, run on agarose gel to visualize correct insert size and orientation.

How is Sequencing confirmation used to screen colonies for correct cloning?

Sequence with vector-specific primers (e.g., T7, SP6, M13); essential to confirm correct sequence, proper orientation, and no PCR-induced mutations.

What are the possible causes if there are no colonies after cloning?

ligase buffer or competent cells expired, incorrect antibiotic concentration, low transformation efficiency

What are the possible causes if there are a lot of blue colonies after cloning?

likely vector self-ligation or uncut vector contamination; use dephosphorylation of vector ends (alkaline phosphatase) to prevent re-ligation.

What are the possible causes if colonies are all white colonies, but no insert is detected after cloning?

primers may have amplified a different or truncated fragment; confirm primer specificity and gel-purify PCR product before cloning.

What is the general overview of the human genome?

About 3 billion base pairs across 23 pairs of chromosomes; contains ~20,000–25,000 protein-coding genes—surprisingly, only ~1.5% of the genome

What are introns?

Non-coding regions within genes.

What are intergenic regions?

Non-coding stretches between genes

What are regulatory sequences?

Promoters, enhancers, silencers

What are repeats?

Satellite DNA, transposons, and retroelements

What are the functions of non-coding DNA?

Regulates gene expression, houses non-coding RNAs, and plays roles in structural and chromosomal organization.

What were the aims of the Human Genome Project (HGP)?

Sequence the entire human genome, identify all human genes, improve technologies for sequencing and bioinformatics, explore genetic variation and its role in disease

What were the key outcomes of the Human Genome Project (HGP)?

Kickstarted precision medicine and systems biology, led to the development of databases like NCBI GenBank, Ensembl, and UCSC Genome Browser, revealed the complexity of gene regulation and the importance of epigenetics.

How does Sanger (dideoxy) sequencing work?

Based on selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) during DNA synthesis; Generates DNA fragments of varying lengths, each ending in a fluorescent ddNTP, fragments separated by capillary electrophoresis, and the sequence is read by a detector.

What are advantages of Sanger sequencing?

high accuracy (~99.99%), excellent for small-scale projects, confirmatory sequencing

What are the limitations of Sanger sequencing?

low throughput, max read length ~800–1000 bp, inefficient for large genomes

How does Microarray technology work?

A chip contains thousands of DNA probes attached in a grid, fluorescently labeled cDNA is hybridized to the chip, signal intensity corresponds to gene expression level.

What are the strengths of using microarrays?

Simultaneous profiling of thousands of genes, established bioinformatics pipelines for clustering and heatmapping

What are the weaknesses of using microarrays?

Limited to known genes, cross-hybridization and signal saturation can reduce accuracy, being replaced in many applications by RNA-Seq, which is more versatile and data-rich

How can Sanger sequencing and microarrays be linked?

Scientists can Confirm gene identity and detect mutations or polymorphisms (Sanger) and Profile how gene expression changes in response to disease, treatments, or environmental factors (microarrays).

What is Next Generation Sequencing (NGS)?

high-throughput sequencing technologies that allow millions to billions of DNA fragments to be sequenced simultaneously, dramatically increasing speed and reducing cost compared to Sanger

How does Library Preparation work in NGS?

Genomic DNA is fragmented, adapters are ligated to each fragment for sequencing and amplification, library fragments may be barcoded for multiplexing.

How does Amplification work in NGS?

Bridge PCR (Illumina): Fragments bind to a flow cell and amplify locally into clusters., Emulsion PCR (Ion Torrent): DNA is captured on beads in oil droplets and amplified individually.

How does sequencing by synthesis work in NGS?

Each base is read as it's added to the growing strand; Fluorescence (Illumina), pH (Ion Torrent), or real-time detection (Nanopore/PacBio).

How does Image/Data Capture & Base Calling work in NGS?

Signals are converted into raw sequences (FASTQ files) with quality scores

How does Bioinformatics Processing work in NGS?

Sequence alignment to reference genome (e.g., using BWA, Bowtie), variant calling (e.g., GATK) and downstream analysis (e.g., visualization, annotation).

What are the strengths and Challenges of Illumina platform for NGS?

High accuracy; industry standard but short reads may miss structural variants

What are the strengths and Challenges of Ion Torrent for NGS?

Fast turnaround; semiconductor-based, but prone to indel errors (homopolymer runs)

What are the strengths and Challenges of PacBio (HiFi) for NGS?

Long reads; ideal for structural insights, but lower throughput, expensive

What are the strengths and Challenges of Oxford Nanopore for NGS?

Portable; ultra-long reads; real-time, but higher error rates; improving rapidly

What does Whole genome sequencing (WGS) do?

Identify rare mutations and structural changes

What does Whole exome sequencing (WES) do?

Focus on protein-coding regions (~1% of genome)