Lecture 2: Recombinant DNA Technology

Flashcards covering key vocabulary from the lecture on Recombinant DNA Technology, including molecular biology concepts, cloning vectors, gene transfer methods, sequencing, and libraries.

Dogma of Molecular Biology

The central concept describing the flow of genetic information: DNA makes RNA, and RNA makes protein.

Recombinant DNA (rDNA) molecules

DNA molecules formed using laboratory methods of genetic recombination to combine material from multiple sources, creating sequences not naturally found in the genome.

Genetic engineering / Molecular cloning

The process of producing many copies of a single fragment of DNA, such as a gene.

Clone

A group of individual cells or organisms descended from one progenitor.

Restriction Enzymes

DNA-cutting enzymes, also called 'molecular scissors,' primarily found in bacteria, that recognize and cut DNA within specific palindromic sequences called restriction sites.

Plasmid DNA Vectors

Circular forms of self-replicating DNA that can be manipulated to carry and clone other pieces of DNA.

Somatostatin

The first human protein made using recombinant DNA technology, known for regulating growth hormone.

Restriction Site

A specific palindromic sequence of bases within a DNA strand that restriction enzymes recognize and cut.

Palindrome (DNA)

A DNA sequence that reads the same forward and backward on opposite DNA strands.

DNA Ligase

An enzyme that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond.

Alkaline Phosphatase

An enzyme that removes 5' phosphate groups from DNA and RNA, most active at alkaline pH.

Origin of Replication (ori)

A site for DNA replication on a plasmid, allowing it to replicate independently from the host chromosome.

Multiple Cloning Site (MCS)

A region within a cloning vector containing recognition sites for several restriction enzymes, where the DNA insert is cloned.

Selectable Marker Genes

Genes (e.g., antibiotic resistance) included in a cloning vector that allow for the selection of transformed cells.

RNA Polymerase Promoter Sequences

Sequences on a cloning vector used for transcription both in vitro and in vivo.

DNA Sequencing Primers

Binding sites on a cloning vector for sequencing primers to verify the inserted DNA.

Bacterial Plasmid Vectors

Cloning vectors capable of cloning DNA inserts up to 10 kb in length.

Bacteriophage Vectors

Cloning vectors that clone DNA inserts up to 25 kb by inserting DNA into a lambda chromosome and packaging it into viral particles.

COS Sites

12 base pair sites at each end of lambda DNA that base pair together when phages infect bacteria, leading to circularization and replication.

Cosmid Vectors

Cloning vectors composed of lambda DNA cos sites, a plasmid origin of replication, and an antibiotic resistance gene, used to clone DNA fragments between 20-40 kb.

Bacterial Artificial Chromosomes (BAC)

Large, low-copy plasmids that can accept DNA inserts ranging from 100-300 kb, historically used in the Human Genome Project.

Yeast Artificial Chromosomes (YAC)

Miniature eukaryotic chromosomes containing an origin of replication, telomeres, selectable markers, and a centromere, best for cloning very large DNA inserts from 200 kb to 2 megabases.

Ti Vectors

Tumor-inducing (Ti) plasmids from pathogenic Agrobacterium species used by scientists to deliver genes to plants, creating transgenic plants.

Transgenic Plants

Plants that have been genetically modified to possess new properties using recombinant DNA technology, often via Ti vectors.

E. coli

A common bacterial host cell used to propagate recombinant DNA molecules due to its easy growth and known genomics.

Saccharomyces cerevisiae (Yeast)

A eukaryotic host cell easily grown with known genomics, used to express and secrete eukaryotic gene products.

Transgenic Animals

Animals whose genetic material has been altered through genetic engineering, often by introducing recombinant DNA.

Electroporation

A physical method for gene transfer into cells using an electrical pulse.

Microinjection

A physical method for gene transfer using fine needles to inject DNA directly into cells.

Liposome Mediated Gene Transfer

A physical method for gene transfer involving DNA encapsulated in lipid vesicles.

Polyethylene Glycol (PEG) Mediated Gene Transfer

A chemical method for gene transfer involving Polyethylene Glycol.

Calcium Chloride Mediated Gene Transfer

A chemical method for gene transfer involving Calcium Chloride.

Whole Genome Shotgun Sequencing

A DNA sequencing method that digests chromosomes into thousands of overlapping fragments (contigs), sequences them, and uses computer programs to align the fragments.

Contigs

Thousands of overlapping DNA fragments produced during whole genome shotgun sequencing that are then aligned to reconstruct the larger sequence.

Genomics

The scientific field focused on the cloning, sequencing, and analyzing of entire genomes.

Next Generation Sequencing (NGS)

A term for various high-throughput DNA sequencing technologies.

Genomic Library

A collection of recombinant DNA clones that collectively represent an organism's entire genome, including both coding (exons) and non-coding (introns) regions.

cDNA Library

A collection of DNA clones representing only the actively expressed genes (exons) of an organism or tissue at a given time, derived from mRNA, and therefore lacking introns.

PCR (Polymerase Chain Reaction)

A technique developed by Kary Mullis for rapidly amplifying a specific sequence of DNA by cycles of denaturation, annealing, and extension.

Denaturation (PCR)

The first stage of PCR where DNA is heated (94-96 °C) to separate its double strands.

Annealing (Hybridization) (PCR)

The second stage of PCR where primers bind to complementary bases at the opposite ends of the target DNA sequence (55-65 °C).

Extension (Elongation) (PCR)

The third stage of PCR where DNA polymerase copies the target DNA (70-75 °C).

Restriction-Modification System

A bacterial defense mechanism where specific DNA sequences in the bacterium's own genome are methylated to prevent its restriction enzymes from cutting them.

Good Cloning Vector

An ideal cloning vector possessing an origin of replication, a multiple cloning site (MCS), and a selectable marker gene.

cDNA Library Source

Tissues or cells specifically expressing the gene of interest under certain conditions to ensure the target mRNA is present and abundant for library construction.