Class 16-24: Molecular Biology Techniques

These flashcards cover key concepts in molecular biology techniques such as PCR, DNA repair mechanisms, genome manipulation, and translation processes.

PCR

Polymerase Chain Reaction; it amplifies a specific DNA sequence in vitro using enzymatic replication.

Template DNA

The DNA that is to be amplified during PCR.

Primers

Short DNA sequences that initiate DNA synthesis in PCR.

dNTPs

Deoxynucleotide triphosphates, the building blocks of DNA used in PCR.

Taq polymerase

Heat-tolerant polymerase used for DNA synthesis in PCR.

Mg²⁺-containing buffer

Buffer that provides magnesium ions, crucial for Taq polymerase activity.

Denaturation

The first step in PCR where the double-stranded DNA template is separated into single strands at ~94°C.

Annealing

The step in PCR where primers bind to the template DNA at a temperature of 30-65°C.

Extension

The step in PCR where DNA polymerase synthesizes new DNA strands at 68-72°C.

5' → 3' synthesis

The direction in which DNA polymerase adds nucleotides to growing DNA strands.

Exponential amplification

Each cycle of PCR doubles the number of target DNA molecules, following a 2ⁿ growth pattern.

ddNTPs

Dideoxynucleotides used in Sanger sequencing that cause chain termination.

Sanger sequencing

A method of DNA sequencing using ddNTPs and gel electrophoresis to read the DNA sequence.

Fluorescently labeled ddNTPs

Modified ddNTPs used in modern Sanger sequencing for simultaneous detection of all bases.

DNA sequences

Written in the 5' → 3' direction, always from left to right.

DNA damage

Alterations to the DNA structure caused by environmental factors such as UV light and chemicals.

Single-nucleotide lesion repair

Repair mechanisms that fix damage to individual nucleotides.

Double-strand break (DSB) repair

Repair mechanisms that fix breaks occurring simultaneously in both strands of the DNA helix.

Mismatch repair

A single-nucleotide lesion repair pathway that corrects base-pairing errors.

Base-excision repair

A pathway that removes and replaces damaged nucleotides in DNA.

Nucleotide-excision repair

A repair mechanism that removes bulky DNA lesions.

Direct repair

A repair process that fixes specific types of DNA damage without excising (removing something completely) nucleotides.

Homologous recombination (HR)

A high-fidelity DNA repair process utilizing a homologous template.

Non-homologous end joining (NHEJ)

An error-prone DNA repair mechanism that directly joins broken ends without a template.

RecA

A protein in bacteria that facilitates homologous recombination.

Rad51

A eukaryotic protein that plays a similar role as RecA in homologous recombination.

D-loop

A displacement loop formed during homologous recombination that allows for DNA synthesis.

Double Holliday junction

A structure formed during double-strand break repair involving two crossed DNA strands.

Crossover

A genetic exchange during homologous recombination that results in mixed parental chromosomes.

Non-crossover

An outcome in homologous recombination where there is no exchange of DNA material between homologs.

Ku70/80 heterodimer

Proteins that initiate non-homologous end joining by binding to DNA ends.

NHEJ in immunity

The role of non-homologous end joining in generating diverse antibodies during VDJ recombination.

DSBR

Double-Strand Break Repair; a process that repairs double-stranded DNA breaks.

Exonucleases

Enzymes that create single-stranded overhangs at double-strand breaks.

Crossover outcomes in DSBR

Determined by the orientation of the endonuclease cuts during Holliday junction resolution.

Aneuploidy

An abnormal number of chromosomes due to improper chromosomal segregation.

Human oocytes and aneuploidy

Oocytes are especially prone due to long periods of homolog connection before meiosis resumes.

Spo11

An enzyme that induces double-strand breaks during meiosis.

Cohesin complexes

Protein structures that stabilize homologous chromosome pairing during meiosis.

Gene disruption using HR

Achieved by introducing a DNA construct with homologous arms for targeted integration.

Positive-Negative selection

A method used in mammalian cells to isolate true homologous recombination events. It differentiates between cells with successful integration of a desired gene and those without, often using selectable markers.

neo and tk markers

Selective markers used in genetic engineering: neo (positive selection)confers drug resistance, and tk (negative selection) confers sensitivity. These markers are used to identify successfully modified cells during the transformation process.

CRISPR/Cas9

A bacterial adaptive immune system adapted for gene editing.

crRNA

CRISPR RNA that guides Cas9 to the specific DNA target.

tracrRNA

Trans-activating crRNA that helps form the active RNA-protein complex with Cas9.

Guided RNA (gRNA)

A fusion of crRNA and tracrRNA designed for precise target recognition.

PAM sequence

Protospacer Adjacent Motif required for Cas9 to recognize and cleave DNA.

CRISPR cuts repair pathway

Primarily via NHEJ creating indels (an insertion or deletion mutation) or precise HR for targeted edits.

Knockout

The result of a CRISPR procedure using non-homologous end joining.

Knock-in

The result of a CRISPR procedure using homologous recombination.

Ribosomes

Molecular machinery for protein synthesis that translates mRNA into amino acids, consisting of rRNA and proteins.

mRNA

Messenger RNA that serves as an intermediate between DNA and protein synthesis.

Adaptor hypothesis

Crick's prediction of tRNAs linking amino acids to specific codons.

tRNA

Transfer RNA that transfers amino acids during the process of translation. It reads the mRNA sequence and ensures the correct amino acid is incorporated into the growing polypeptide chain.

UUU codon

A specific RNA triplet encoding the amino acid phenylalanine. It is found in messenger RNA and is also known as a start codon in some contexts.

The genetic code

A set of rules defining how nucleotide sequences translate into amino acids.

Universal genetic code

The consistency of codon-amino acid relationships across nearly all organisms.

DNA-dependent RNA polymerase

The enzyme that synthesizes RNA by transcribing from a DNA template.

Holoenzyme

The complete functional form of RNA polymerase, consisting of a core enzyme and a sigma factor.

Sigma factor

A protein that identifies promoter sequences for RNA polymerase initiation.

Consensus promoter sequences

Common motifs found in bacterial promoters recognized by sigma factors.

Transcription direction

Transcription proceeds toward the 3′ end of the template strand.

Open complex

The unwound region of DNA where transcription initiation occurs.

Abortive initiation

The process where RNA polymerase synthesizes short RNA fragments before elongation.

Scrunching model

A mechanism describing how RNA polymerase stores energy during abortive initiation.

Elongation phase

The phase where RNA polymerase synthesizes RNA after sigma release.

DNA gyrase

An enzyme that alleviates the tension in DNA ahead of the transcription bubble.

FRET

Fluorescence resonance energy transfer, used to demonstrate the scrunching model during transcription.

Replication, transcription, and PCR

All processes that rely on base-pair complementarity and 5'→3' polymerization.

HR, DSBR, and CRISPR

Molecular mechanisms involved in creating and repairing double-strand breaks.

High fidelity HR vs error-prone NHEJ

HR uses a homologous template for accuracy, whereas NHEJ relies on blunt end joining.

Translation experiments and the central dogma

Demonstrated the link between DNA, RNA, and protein synthesis.

Recombination and CRISPR

Both rely on precise molecular recognition to manipulate DNA sequences.

Chemical machines of life

Enzymatic systems like polymerases and ribosomes sustaining biological life.