Chapter 10: Analyzing the Structure and Function of Genes

Flashcards for reviewing key vocabulary and concepts from Chapter 10 Lecture Notes on Analyzing the Structure and Function of Genes.

Selective breeding

A form of genetic manipulation where humans have manipulated DNA without knowing what they are doing.

DNA cloning

The production of many identical copies of a DNA sequence.

Restriction enzymes

Enzymes that cleave both strands of the DNA double helix at specific nucleotide sequences, discovered in bacterial cells to degrade foreign DNA.

Blunt ends

Result from restriction enzyme cleavage where both DNA strands are cut at the same position, leaving no overhangs.

Sticky ends

Result from restriction enzyme cleavage where the cut produces an overhang, often used to join DNA fragments.

Gel electrophoresis

A technique used to separate DNA fragments based on size, where negatively charged DNA migrates toward a positive electrode.

DNA ligase

An enzyme that can join together any two DNA fragments in vitro by sealing nicks between the phosphodiester backbone, producing recombinant DNA molecules.

Recombinant DNA

DNA fragments from different sources joined together, typically using DNA ligase, for cloning.

Plasmids

Small, circular DNA molecules commonly used as cloning vectors, containing an origin of replication allowing independent replication.

Cloning vectors

Small, circular DNA molecules (like plasmids) used to carry DNA to be cloned inside other cells.

Origin of replication

A specific sequence in plasmids that allows them to replicate independently of the host genome.

Transformation

The process by which bacteria naturally take up DNA from their surroundings, leveraged in the lab for DNA cloning.

DNA library

A collection of cloned DNA fragments in a bacterial culture.

Genomic library

A collection of cloned DNA that represents the entire genome of a cell.

Complementary DNA (cDNA)

DNA fragments synthesized from mRNA using reverse transcriptase, advantageous for containing only coding sequences without introns.

cDNA library

A collection of DNA fragments synthesized using all mRNA in a cell, which will differ cell to cell and can change during various cell stages.

DNA denaturation

The process of unwinding DNA at high temperatures (~90˚C) to separate its complementary strands.

DNA renaturation (hybridization)

The process where denatured DNA strands reform hydrogen bonds between complementary base pairs as they cool.

Polymerase chain reaction (PCR)

A technique for amplifying select DNA by multiple cycles of DNA synthesis in a test tube, making thousands of copies quickly.

PCR primers

Short synthetic DNA sequences that hybridize to specific regions of the template DNA to direct amplification in PCR.

DNA fingerprint

A unique profile of DNA variations used to identify a person, often collected using PCR due to its sensitivity.

Dideoxy method (Sanger sequencing)

A method for determining the nucleotide sequence of DNA that utilizes DNA polymerase and chain-terminating dideoxynucleoside triphosphates (ddNTPs).

Dideoxynucleoside triphosphates (ddNTPs)

Modified nucleotides used in Sanger sequencing that terminate DNA synthesis when incorporated, as they lack a 3'-OH group.

Shotgun sequencing

A method for sequencing entire genomes by breaking the genome into fragments, sequencing them, and then reassembling the sequence.

Clone-by-clone approach

A method of genome sequencing that breaks the genome into overlapping fragments, plugs them into BACs, and inserts them into E. coli for mapping and sequencing.

Bacterial artificial chromosomes (BACs)

Cloning vectors capable of carrying large fragments (100-200 kilobase pairs) of DNA, used in the clone-by-clone sequencing approach.

Illumina sequencing

A high-throughput sequencing method based on principles similar to automated dideoxy sequencing, using removable fluorescently labeled chain-terminating nucleotides.

RNA sequencing (RNA-seq)

A sequencing technique used to determine the nucleotide sequence of a collection of RNAs, allowing analysis of a cell’s transcriptome.

Transcriptome

The complete set of RNA transcripts produced by the genome under specific conditions.

In situ hybridization

A technique using a labeled ssRNA or DNA probe to locate a complementary nucleotide sequence in a chromosome, cell, or tissue.

Ribosome profiling

A method that reveals which mRNAs are translated by protecting ribosome-bound mRNA fragments from RNase digestion, which are then converted to cDNA and sequenced.

Reporter genes

Genes encoding a protein whose activity is easy to monitor experimentally (e.g., by fluorescence or enzymatic activity) to study gene expression patterns.

Green fluorescent protein (GFP)

A highly fluorescent protein widely used experimentally as a reporter gene to identify specific cells and monitor gene expression in living organisms.

RNA interference (RNAi)

A popular method for silencing genes to study their function, where double-stranded RNA (dsRNA) destroys matching nucleotide sequences in the cell.

dsRNA

Double-stranded RNA; in RNAi, it is cleaved into siRNAs that direct the degradation of target mRNAs.

siRNAs

Short interfering RNAs, produced from dsRNA by RNAi machinery, which hybridize with target gene's mRNAs and direct their degradation.

Transgenic organisms (GMOs)

Plants or animals that have stably incorporated one or more genes derived from another cell or organism into their genome.

Targeted gene replacement

A technique, often utilizing embryonic stem cells in mice, to introduce mutant genes into an organism for analysis of gene activity.

Gene knockouts

Organisms created by introducing two inactive versions, mutant versions, or deleting a gene altogether, often to study gene function.

Conditional knockouts

A technique that allows the selective disruption of a gene in a particular cell type or at a certain stage in development.

CRISPR system

A gene editing system based on bacterial enzymes like Cas9 that produces double-stranded DNA breaks at target sequences directed by guide RNAs.

Cas9

A bacterial enzyme used in the CRISPR gene editing system that produces double-stranded DNA breaks.

Guide RNAs

RNA molecules used in the CRISPR system to direct Cas9 to a specific target DNA sequence.

Callus

A mass of undifferentiated plant cells that can proliferate indefinitely in a disorganized manner when plant tissue is cultured in a sterile medium.

Expression vectors

Vectors that include transcription and translation signals to direct the expression of a gene at high levels in host cells, enabling production of large amounts of protein.