Genomics and Genetic Engineering Concepts

These flashcards encompass key vocabulary and concepts from the lecture series on genomics, genetic engineering, DNA sequencing, and gene regulation.

DNA strand extension

The enzymatic process where DNA polymerase adds complementary nucleotides, one by one, to the 3'-hydroxyl end of a growing DNA strand, following the sequence information provided by a template strand.

DNA polymerase

A crucial enzyme responsible for synthesizing new DNA strands. It catalyzes the addition of deoxynucleotides (dNTPs) that are complementary to those on a template DNA strand, extending the new DNA chain in the 5' to 3' direction. It also typically possesses proofreading activity.

Phosphodiester bond

A strong covalent bond that forms the backbone of DNA and RNA molecules. It links the 3'-hydroxyl group of one nucleotide's sugar to the 5'-phosphate group of the next nucleotide, creating a continuous sugar-phosphate chain.

Restriction enzymes

Specialized bacterial enzymes that recognize and cleave phosphodiester bonds at specific nucleotide sequences, known as recognition sites, within DNA molecules. They produce DNA fragments with either blunt or sticky ends and are indispensable tools in molecular cloning and genetic engineering.

Blunt ends

The ends of DNA fragments that result from restriction enzyme cleavage where both DNA strands terminate in a base pair, lacking any single-stranded overhangs. Joining these fragments through ligation is generally less efficient due to the absence of complementary base-pairing to guide the process.

Sticky ends

The ends of DNA fragments generated by certain restriction enzymes, characterized by short, single-stranded overhangs. These overhangs are complementary to other sticky ends created by the same enzyme, allowing them to anneal via hydrogen bonds and facilitating efficient ligation, making them ideal for molecular cloning.

DNA ligase

An essential enzyme that catalyzes the formation of a phosphodiester bond between the 3'-hydroxyl end of one nucleotide and the 5'-phosphate end of an adjacent nucleotide. It is critical for repairing DNA nicks, joining Okazaki fragments during DNA replication, and sealing DNA fragments during recombinant DNA procedures.

Plasmid vector

A small, circular, extrachromosomal DNA molecule, typically found in bacteria, that can autonomously replicate and is engineered to carry foreign DNA fragments into host cells. Plasmids are commonly used as vectors in genetic engineering for gene cloning, expression, and transfer.

Recombinant DNA

A hybrid DNA molecule created in vitro by joining DNA fragments from two or more different biological sources. This technology is fundamental to genetic engineering, enabling the combination of genetic material in novel ways to produce altered organisms or proteins.

Sanger sequencing

A pioneering method for determining the precise order of nucleotides (A, T, C, G) in a DNA molecule. It relies on in vitro DNA synthesis, where the incorporation of chain-terminating dideoxynucleotides (ddNTPs) at random positions generates DNA fragments of varying lengths, which are then separated by size.

dideoxynucleotide (ddNTP)

A modified nucleotide used in Sanger sequencing that lacks a hydroxyl (-OH) group at both the 2' and 3' carbons of its deoxyribose sugar. When a ddNTP is incorporated into a growing DNA strand during synthesis, it prevents the addition of further nucleotides, thereby terminating the elongation of the DNA chain.

deoxynucleotide (dNTP)

A standard nucleotide, consisting of a deoxyribose sugar, a phosphate group, and one of four nitrogenous bases (adenine, guanine, cytosine, or thymine). dNTPs are the basic building blocks used by DNA polymerase to synthesize new DNA strands.

Primer

A short, single-stranded nucleic acid sequence (either DNA or RNA) that provides a free 3'-hydroxyl group. This 3'-OH group serves as the starting point for DNA polymerase to begin synthesizing a new complementary DNA strand during DNA replication or PCR.

Gel/capillary electrophoresis

A laboratory technique used to separate macromolecules, such as DNA fragments or proteins, based on their size and charge. In the context of DNA, fragments migrate through a gel matrix (or capillary) under the influence of an electric field, with smaller fragments moving faster and further.

Genomic library

A comprehensive collection of cloned DNA fragments that collectively represent the entire genome of a specific organism. Each fragment is typically inserted into a vector (like a plasmid, cosmid, or bacteriophage) and stored in a host cell, allowing researchers to isolate and study individual genes or genomic regions.

Clone

In molecular biology, a gene or DNA fragment that has been inserted into a vector and replicated numerous times within a host cell, producing many identical copies for further study, sequencing, or manipulation.

Paired-end reads

A sequencing strategy where both ends of a single DNA fragment of a known size are sequenced. This technique generates two sequence reads (one from each end) that are separated by a consistent distance, which is invaluable for genome assembly, detecting rearrangements, and mapping reads more accurately.

Contig

A contiguous stretch of DNA sequence that is computationally assembled by overlapping shorter, individual sequencing reads. Contigs represent a merged consensus sequence for a given genomic region and are crucial for reconstructing larger DNA sequences, such as entire chromosomes, from fragmented sequencing data.

Scaffold

An ordered series of contigs that are linked together by paired-end read information, separated by gaps of unknown sequence. Scaffolds provide a higher-level assembly of a genome, indicating the relative order and orientation of contigs, even if the intervening sequence is not yet determined.

Sequence walking

A method used to extend a DNA sequence from a known region into adjacent, unknown regions. It involves designing new primers based on the sequence determined in the previous step and then using PCR or sequencing to 'walk' further along the chromosome, effectively filling gaps in a genome assembly.

Transposon

Also known as 'jumping genes,' these are segments of DNA that have the ability to move from one location to another within a genome, either by direct excision and reinsertion (cut-and-paste) or by replication through an RNA intermediate (copy-and-paste). They can cause mutations, alter gene regulation, and contribute to genome evolution.

DNA transposon

A class of transposons that move within the genome via a 'cut-and-paste' mechanism. The transposase enzyme, encoded by the transposon itself, excises the DNA element from its original genomic location and inserts it into a new, often random, site. This process typically leaves the copy number of the transposon unchanged.

RNA transposon (retrotransposon)

A class of transposable elements that move via an RNA intermediate, hence often called 'retrotransposons.' They are first transcribed into an RNA molecule, which is then reverse-transcribed into a new DNA copy by reverse transcriptase. This new DNA copy is subsequently inserted into a different genomic location, leading to an increase in the transposon's copy number within the genome.

LINE/SINE

Long Interspersed Nuclear Elements (LINEs) and Short Interspersed Nuclear Elements (SINEs) are two major types of non-LTR retrotransposons in mammalian genomes. LINEs are autonomous, encoding reverse transcriptase, while SINEs are non-autonomous, relying on LINE-encoded machinery for their transposition. They are significant contributors to genome size and evolution.

Illumina sequencing

A widely used high-throughput sequencing platform (also known as Sequencing by Synthesis) that generates millions of short, highly accurate DNA sequence reads in parallel. It is characterized by its high output, low error rate, and suitability for applications such as whole-genome sequencing, RNA-seq, and ChIP-seq.

PacBio sequencing

A long-read sequencing technology (Pacific Biosciences) that allows for single-molecule, real-time sequencing. It produces significantly longer reads (up to tens of thousands of base pairs) compared to Illumina, which is beneficial for resolving repetitive regions and complex genome assemblies, albeit with a typically higher raw error rate.

Oxford Nanopore sequencing

A novel, portable, and ultra-long-read sequencing technology that works by passing individual DNA or RNA strands through a protein nanopore. The changes in electrical current as nucleotides traverse the pore are detected and translated into a sequence. It offers real-time data and can generate reads hundreds of kilobases long, making it useful for rapid sequencing and field applications.

Genome annotation

The computational process of identifying all the functional elements within a sequenced genome. This includes locating protein-coding genes, non-coding RNA genes, regulatory sequences (like promoters and enhancers), repetitive elements, and predicting their biological functions. It transforms raw sequence data into biologically meaningful information.

Codon

A sequence of three consecutive nucleotides in a messenger RNA (mRNA) molecule that specifies a particular amino acid during protein synthesis (translation), or signals for the termination of translation. The genetic code defines which specific codons correspond to each of the 20 amino acids.

Open reading frame (ORF)

A continuous stretch of DNA or RNA sequence that begins with a start codon (typically AUG/ATG) and ends with a stop codon (UAA, UAG, UGA/TAA, TAG, TGA) without any intervening stop codons. ORFs are indicative regions that have the potential to encode a protein or polypeptide.

Conserved sequence

A DNA or RNA sequence that has remained largely unchanged or identical across different species or within different genes of the same species over evolutionary time. Such high levels of conservation often indicate that the sequence plays a critical functional role (e.g., in gene regulation or protein coding) and is under strong purifying selection.

RNA-seq

A powerful, high-throughput sequencing technology used to comprehensively analyze the transcriptome — the complete set of RNA transcripts present in a cell or tissue at a given moment. It quantifies gene expression levels, identifies novel transcripts, alternative splicing events, and RNA editing, providing a global view of cellular gene activity.

cDNA library

A collection of cloned complementary DNA (cDNA) molecules, each reverse-transcribed from a messenger RNA (mRNA) molecule isolated from a specific cell type, tissue, or developmental stage. Unlike genomic libraries, cDNA libraries only contain sequences of actively expressed genes (exons), making them useful for studying gene expression and identifying transcribed sequences.

Expressed sequence tag (EST)

A short, single-read cDNA sequence, typically generated from one end of a cDNA clone. ESTs serve as molecular markers for identifying actively transcribed genes (expressed genes) in an organism and are useful for gene discovery, mapping, and understanding gene expression patterns.

Chromosomal rearrangements

Large-scale structural changes in the organization of chromosomes, which can involve the gain, loss, or relocation of entire segments of DNA. Common types include translocations (exchange between non-homologous chromosomes), inversions (segment flipped), deletions (segment lost), and duplications (segment copied), often leading to significant genetic and phenotypic consequences.

Karyotyping

A cytogenetic technique used to visually analyze the complete set of chromosomes in a cell. Chromosomes are stained, arranged in pairs, and examined under a microscope to detect large-scale numerical or structural abnormalities, such as aneuploidy, translocations, or large deletions/duplications.

FISH

Fluorescence In Situ Hybridization (FISH) is a molecular cytogenetic technique that uses fluorescently labeled DNA or RNA probes to specifically bind to complementary target nucleic acid sequences within cells or chromosomes. This allows for the precise visualization and localization of specific genes, chromosomal regions, or mRNA molecules under a fluorescence microscope.

Comparative Genomic Hybridization (CGH)

A molecular cytogenetic technique used to detect DNA copy number variations (gains or losses of genetic material) across the entire genome. It involves co-hybridizing differentially labeled 'test' and 'reference' genomic DNA to normal metaphase chromosomes or a microarray, revealing regions of imbalance.

PCR / Southern blot

PCR (Polymerase Chain Reaction) is a molecular biology technique used to amplify a specific DNA sequence exponentially. Southern blot is a laboratory method used to detect specific DNA sequences within a DNA sample, identifying DNA fragments of interest after separation by gel electrophoresis. Both can be used to detect specific DNA sequences or rearrangements, with PCR being faster for targeted amplification and Southern blot for detecting larger structural changes or copy number variations.

Promoter

A specific DNA sequence region located immediately upstream (5') of a gene. It serves as the binding site for RNA polymerase and various general transcription factors, directing the initiation of gene transcription and playing a critical role in regulating when and how frequently a gene is transcribed.

Enhancer

A regulatory DNA sequence that significantly boosts the transcription rate of its target gene, often by binding specific activator proteins. Enhancers can function effectively over long distances (thousands of base pairs away), either upstream, downstream, or even within introns of the gene they regulate, typically through DNA looping.

Activator

A type of transcription factor protein that binds to specific DNA sequences, such as enhancers or upstream promoter elements, to increase (activate) the transcription of a gene. Activators facilitate the recruitment of RNA polymerase or general transcription factors to the promoter, thereby stimulating gene expression.

Repressor

A type of transcription factor protein that binds to specific DNA sequences (e.g., operator sequences, silencers, or promoter regions) to decrease or prevent the transcription of a gene. Repressors exert their inhibitory effects by physically blocking RNA polymerase's access to the promoter or by hindering the binding of activators.

Transcription factor (TF)

A protein that regulates gene expression by binding to specific DNA sequences, typically within or near a gene's promoter, enhancer, or silencer regions. Transcription factors can either activate (activators) or repress (repressors) the rate of gene transcription, thereby controlling which genes are turned on or off in a cell.

Basal transcription factors

A set of essential proteins that assemble at the core promoter of most eukaryotic protein-coding genes to form the preinitiation complex (PIC). They are required for recruiting RNA polymerase II and establishing a minimal, low level of transcription, even in the absence of specific regulatory activators.

Reporter assay

An experimental molecular biology technique used to measure the transcriptional activity of a promoter or enhancer. The regulatory DNA sequence of interest is fused to a 'reporter gene' (e.g., encoding eta-galactosidase (lacZ) or Green Fluorescent Protein (GFP)), whose expression can be easily detected and quantified. The level of reporter gene activity then reflects the strength of the regulatory sequence.

ATAC-seq

Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) is a molecular biology technique used to map regions of 'open' or accessible chromatin across the genome. It utilizes a hyperactive transposase enzyme to cut and tag DNA at accessible sites, providing insights into active regulatory elements like promoters and enhancers.

Transcription factor binding motif

A short, specific DNA sequence (typically 6-12 base pairs long) that is recognized and bound by a particular transcription factor protein. These motifs are often degenerate, meaning there can be slight variations in the sequence while still allowing for binding, and are critical for transcriptional regulation.

ChIP-seq

Chromatin Immunoprecipitation sequencing (ChIP-seq) is a powerful molecular biology technique used to map the genome-wide locations where specific DNA-binding proteins (e.g., transcription factors, histones, or modified histones) interact with DNA. It involves chemically crosslinking proteins to DNA, fragmenting the chromatin, immunoprecipitating the protein-DNA complexes with specific antibodies, and then sequencing the enriched DNA.

Nucleosome

The fundamental repeating structural unit of chromatin in eukaryotic cells. Each nucleosome consists of a segment of DNA (approximately 147 base pairs) wrapped extasciitilde1.65 times around an octamer of eight histone proteins (two copies each of H2A, H2B, H3, and H4). Nucleosomes compact DNA and play a key role in gene regulation.

Histone modifications

Reversible chemical changes that occur on the N-terminal tails of histone proteins, such as methylation, acetylation, phosphorylation, and ubiquitination. These modifications alter the charge and shape of histones, influencing how tightly DNA is wrapped around them, thereby regulating chromatin accessibility and gene expression.

Reader/Writer/Eraser

A conceptual framework describing the enzymes and proteins involved in epigenetic regulation. 'Writers' (e.g., histone acetyltransferases, DNA methyltransferases) add chemical modifications to histones or DNA. 'Erasers' (e.g., histone deacetylases, demethylases) remove these modifications. 'Readers' (e.g., proteins with bromodomains or chromodomains) recognize and bind to specific modifications, translating the epigenetic marks into downstream cellular responses, often affecting gene expression.

Forward genetics

A classical genetic research approach that begins by identifying individuals with an observable mutant phenotype (a specific trait or disease). Researchers then work backward to pinpoint the gene(s) responsible for that phenotype, often through techniques like mutagenesis, genetic crosses, and mapping, to understand how genes influence traits.

Reverse genetics

A molecular biology research approach that starts with a known gene sequence. Researchers then systematically alter the function of that gene (e.g., by knockout, knockdown, or overexpression) in a model organism to observe the resulting changes in phenotype. This method is used to deduce the normal biological function of a specific gene.

Saturated genetic screen

A systematic forward genetics approach where a population of organisms is subjected to mutagenesis until all possible mutable genes or loci in the genome are theoretically represented by at least one mutation. The goal is to identify all genes contributing to a specific biological process or phenotype.

Quantitative trait locus (QTL)

A region on a chromosome that is statistically associated with the variation of a quantitative trait (a trait that shows continuous variation, like height or weight). QTL mapping uses genetic markers to identify these genomic regions, which often contain multiple genes contributing to the trait.

Quantitative traits

Complex biological characteristics that exhibit continuous variation within a population (e.g., human height, crop yield, blood pressure). These traits are typically influenced by the combined effects of multiple genes (polygenic inheritance) and significant environmental factors. They can be continuous (e.g., height), meristic (e.g., number of bristles), or threshold (e.g., disease susceptibility).

GWAS

Genome-Wide Association Study (GWAS) is a research approach that rapidly scans the entire genomes of large cohorts of individuals to identify genetic variants, primarily Single Nucleotide Polymorphisms (SNPs), that are statistically associated with a particular disease or observable trait. It is a powerful tool for discovering genetic contributions to common, complex traits.

Linkage disequilibrium (LD)

The non-random association of alleles at two or more different loci within a population, meaning that specific combinations of alleles occur together on a chromosome more frequently than would be expected by chance. LD is often observed between physically close genes and is a fundamental principle used in genetic mapping and GWAS to locate disease genes.

Housekeeping gene

A gene that is constitutively expressed (continuously active) at a relatively constant level in virtually all cells of an organism, regardless of cell type, developmental stage, or environmental conditions. The proteins encoded by housekeeping genes are essential for fundamental cellular functions necessary for cell viability and basic metabolism.

Tissue-specific gene

A gene whose expression is limited to or significantly enriched in particular cell types or tissues within an organism. The precise regulation of tissue-specific genes, often by specialized enhancers and transcription factors, is critical for establishing and maintaining cell identity and specialized functions.

Insulator

A specific DNA sequence element that functions as a boundary to regulate gene expression by preventing inappropriate interactions between regulatory elements. Insulators can block the spread of heterochromatin or prevent an enhancer from acting on a gene outside its designated chromatin domain, thus defining independent transcriptional units.

DNA looping

A fundamental mechanism in genome organization and gene regulation where distant DNA sequences, such as enhancers and promoters, are brought into close physical proximity through the formation of chromatin loops. This allows for direct protein-protein interactions between regulatory factors bound at distant sites, facilitating transcriptional control.

Cohesin/CTCF

Cohesin is a multi-subunit protein complex that encircles DNA, primarily known for holding sister chromatids together during cell division. CTCF (CCCTC-binding factor) is a highly conserved DNA-binding protein. Together, Cohesin and CTCF play crucial roles in organizing the 3D chromatin architecture by mediating the formation of chromatin loops and acting as

Loop extrusion model

A widely accepted model explaining how chromatin loops are formed and maintained in the nucleus. It proposes that cohesin complexes actively extrude DNA through their ring-like structure, creating a loop, until they encounter boundary elements like CTCF proteins, which block further extrusion and define the loop's size and position.

Double-stranded break (DSB)

A severe form of DNA damage characterized by a complete break in both phosphodiester backbones of the DNA double helix. DSBs are highly cytotoxic and must be repaired efficiently to maintain genome integrity. They are also intentionally induced in genome editing technologies like CRISPR-Cas9.

Zinc Finger Nucleases (ZFNs)

Engineered proteins that facilitate targeted genome editing by creating site-specific double-stranded breaks in DNA. They consist of a DNA-binding domain (composed of custom-designed zinc finger motifs that recognize specific DNA triplets) fused to a nuclease domain (typically the FokI cleavage domain), allowing for precision cutting at chosen genomic loci.

TALENs

Transcription Activator-Like Effector Nucleases (TALENs) are engineered proteins used for targeted genome editing. They are composed of a DNA-binding domain (derived from Transcription Activator-Like Effector proteins, where each module recognizes a single specific nucleotide) fused to a nuclease domain (usually FokI). TALENs can be highly specific and are designed to cut DNA at desired sequences.

CRISPR-Cas9

A revolutionary genome-editing technology derived from a bacterial adaptive immune system. It utilizes a guide RNA (gRNA) molecule to precisely direct the Cas9 nuclease enzyme to a specific target DNA sequence, where Cas9 acts as molecular scissors to create a double-stranded break, enabling highly precise and efficient gene editing.

Guide RNA (gRNA)

A synthetic RNA molecule engineered to direct the Cas9 nuclease to a specific target sequence in the DNA genome during CRISPR-Cas9 genome editing. It consists of a spacer sequence (complementary to the target DNA) and a scaffold sequence (which binds to Cas9).

PAM sequence

Protospacer Adjacent Motif (PAM) is a short, specific DNA nucleotide sequence (typically 2-6 base pairs long) that is absolutely required for the Cas9 nuclease to bind to and cleave its target DNA. The PAM sequence is adjacent to the target sequence and acts as a critical recognition signal that ensures Cas9 selectively cuts foreign DNA, not the bacterial host's own DNA.

Non-homologous end joining (NHEJ)

A major and often error-prone DNA repair pathway that re-joins double-stranded breaks (DSBs) in DNA. NHEJ directly ligates the broken DNA ends without requiring a homologous template. This process can lead to small insertions or deletions (indels) at the repair site, potentially resulting in frameshifts or gene knockouts.

Homology-directed repair (HDR)

A high-fidelity DNA repair pathway that accurately repairs double-stranded breaks (DSBs) or other DNA lesions by using a homologous DNA sequence as a template. HDR is typically active during the S and G2 phases of the cell cycle and is widely utilized in advanced gene editing techniques (e.g., CRISPR-Cas9) to introduce specific, precise genetic modifications.

AAV vector

Adeno-Associated Virus (AAV) is a small, non-enveloped virus commonly used as a viral vector in gene therapy due to its low immunogenicity, broad tropism (ability to infect various cell types), and ability to transduce both dividing and non-dividing cells. AAV vectors are engineered to deliver therapeutic genes safely into target cells to treat genetic diseases.

Ancient DNA (aDNA)

DNA extracted from ancient biological samples, such as fossilized bones, teeth, hair, seeds, or museum specimens, spanning from hundreds to hundreds of thousands of years old. aDNA is typically highly degraded, fragmented, contaminated, and chemically modified, making its extraction and analysis challenging but providing invaluable insights into evolutionary history and past populations.

Neanderthals

An extinct species or subspecies of archaic humans that inhabited Eurasia from approximately 400,000 to 40,000 years ago. Genetic studies of ancient DNA have shown that Neanderthals interbred with anatomically modern humans, contributing a small but significant percentage (around 1-4%) of their genome to present-day non-African human populations.

Denisovans

An extinct species or subspecies of archaic humans identified primarily through ancient DNA analysis from fossil fragments found in Denisova Cave in Siberia. Genetic evidence indicates that Denisovans interbred with early modern humans, and their genetic contributions (up to 4-6%) are found in some modern human populations, particularly in Melanesia and Southeast Asia.

In situ hybridization (ISH)

A powerful molecular biology technique used to precisely localize and visualize specific nucleic acid sequences (DNA or RNA) within morphologically preserved cells, tissues, or whole embryos. It employs labeled probes that are designed to bind specifically and complementarily to the target sequence, allowing its spatial distribution to be observed.

Immunolocalization

A laboratory technique used to pinpoint the precise subcellular location of specific proteins within cells or tissues. It involves using antibodies that specifically bind to the target protein. These antibodies are typically conjugated with a fluorescent dye or an enzyme that produces a visible reaction product, allowing the protein's presence and distribution to be visualized under a microscope.

Sufficiency

A concept in biology and genetics referring to whether a particular factor or gene, when introduced or expressed on its own, is enough to produce a specific biological outcome, phenotype, or developmental process, even in the absence of other potentially contributing factors.

Necessity

A concept in biology and genetics referring to whether a particular factor or gene is absolutely required for a specific biological outcome, phenotype, or developmental process to occur. If the factor is necessary, its removal or inactivation would prevent the outcome from happening.

Pax6

A highly conserved transcription factor gene that serves as a master control gene for eye development across diverse animal species, from insects to humans. Its expression is both necessary and sufficient for the initiation of eye formation, highlighting its fundamental role in orchestrating a complex developmental program.

GAL4/UAS system

A powerful binary gene expression system widely used in genetic model organisms, particularly Drosophila, for precise spatial and temporal control of gene expression. The GAL4 protein (transcription activator) derived from yeast binds to upstream activating sequences (UAS) to drive expression of a target gene placed downstream of the UAS. By restricting GAL4 expression to specific cells, researchers can activate target genes only in those cells, allowing for targeted manipulation of gene function.