MOL100H23 lecture 13-mol gen tech (1)

Genotype

The DNA sequence of the genome, specifically referring to the DNA sequence of the two alleles of a gene.

Phenotype

The observable features of an organism or a cell, such as the shape or size of an organ or organelle.

Alleles

The two copies of a gene, each placed on one of the two homologous chromosomes.

Heterozygous

When an organism or cell has one allele carrying a mutation and one non-mutant allele.

Homozygous

When an organism or cell has both alleles carrying the same mutation.

Recessive mutations

Mutations that can only cause a change in phenotype when both alleles carry the mutation.

Dominant mutations

Mutations that can cause a change in phenotype even when only one allele carries the mutation.

Point mutations

Mutations that affect a single nucleotide, such as changing it to a different nucleotide, deletion, or insertion of one nucleotide.

Missense mutations

Point mutations that lead to a change in the encoded amino acid.

Nonsense mutations

Point mutations that generate a stop codon.

Silent mutations

Point mutations that do not change the encoded amino acid.

Frameshift mutations

Mutations caused by insertions or deletions that lead to a shift in the reading frame of codons, often resulting in a stop codon.

Forward genetics

An experimental approach that involves the random generation of mutations and screening the offspring for phenotypes to identify the genotype.

Reverse genetics

An experimental approach that uses molecular techniques to mutate a specific gene and then identifies the resulting phenotype.

In vitro

Outside the normal biological context, such as in cell culture or with purified proteins in a reaction vial.

In vivo

In the biological context, within the organism.

Molecular cloning

A technique that uses specialized enzymes to manipulate DNA for the purpose of studying gene function.

In situ hybridization

A technique used to detect the presence and location of specific mRNA molecules in cells or tissues.

Antisense probe

A single RNA strand that is complementary to a specific mRNA sequence and binds to it with high affinity and specificity.

Complementary DNA (cDNA)

DNA synthesized from mRNA using reverse transcription, which only includes the exons of the gene.

Reverse transcriptase

An enzyme derived from viruses that can synthesize DNA from RNA templates.

Polymerase chain reaction (PCR)

A technique used to amplify specific DNA sequences using DNA polymerase and primers.

DNA polymerase

An enzyme that synthesizes DNA in the 5' to 3' direction and requires primers to start the synthesis.

cDNA

Complementary DNA, a form of DNA synthesized from a messenger RNA (mRNA) template through the action of reverse transcriptase.

DNA amplification

The process of making multiple copies of a specific DNA sequence using techniques such as polymerase chain reaction (PCR).

Polymerase chain reaction (PCR)

A laboratory technique used to amplify a specific DNA sequence by repeatedly heating and cooling the DNA to separate and replicate its strands.

DNA polymerase

An enzyme that synthesizes DNA molecules by adding nucleotides to a growing DNA strand.

Primers

Short DNA sequences that serve as starting points for DNA synthesis in PCR. They are complementary to the target DNA sequence and provide a template for DNA polymerase to bind and initiate synthesis.

Denaturation

The process of separating the double-stranded DNA into single strands by exposing it to high temperatures.

Annealing

The process of binding the primers to their complementary sequences on the single-stranded DNA template.

Elongation

The phase of PCR where DNA polymerase synthesizes new DNA strands using the primers as templates.

Plasmid vector

Small circular DNA molecules found in bacteria that can be used to carry and replicate foreign DNA fragments.

Restriction enzymes

Enzymes that recognize specific DNA sequences and cut the DNA at those sites. They are used in molecular biology to generate compatible ends for DNA ligation.

Transformation

The process of introducing foreign DNA into bacterial cells, allowing them to take up and replicate the DNA.

In situ hybridization

A technique used to detect and localize specific nucleic acid sequences within intact cells or tissues.

Recombinant plasmid

A plasmid that contains a foreign DNA fragment inserted into its DNA sequence.

Transfection

The process of introducing foreign DNA into animal cells, either transiently or stably.

Forward genetics

A genetic approach that involves identifying phenotypes first and then determining the underlying genes responsible for those phenotypes.

Reverse genetics

A genetic approach that involves mutating a specific gene and then studying the resulting phenotype.

RNA interference

A process in which small RNA molecules inhibit gene expression by targeting and degrading specific mRNA molecules.

CRISPR/Cas9 system

A revolutionary gene-editing tool that uses a guide RNA and the Cas9 enzyme to introduce precise changes in the DNA sequence.

Gene targeting by homologous recombination

The method of replacing a gene of interest with an artificial DNA construct using homologous recombination.

Homology arms

Identical sequences in a gene targeting construct that are used to target a specific genomic region.

Chimaeric mouse

A mouse made from cells of different origins, including manipulated embryonic stem cells.

RNA interference

A cellular mechanism that induces the degradation of targeted mRNA.

Dicer

A protein that cleaves long double-stranded RNA molecules into short double-stranded RNAs.

RISC

RNA-induced silencing complex, a protein complex that binds to short double-stranded RNAs and degrades the sense strand.

CRISPR/Cas9 system

A bacterial defense system against phages that can be used for gene manipulation.

CRISPR

Clustered Regularly Interspersed Short Palindromic Repeats, a part of the bacterial CRISPR/Cas system.

Cas genes

Genes associated with the CRISPR/Cas system.

Guide RNA (gRNA)

Engineered RNA that directs Cas9 to the targeted sequence in the genome.

NHEJ

Nonhomologous end joining, a common DNA repair mechanism that is error-prone and can cause small insertions or deletions.

HDR

Homology-directed repair, a DNA repair mechanism stimulated by providing an artificial DNA construct with homology arms.