Comprehensive Guide to Recombinant DNA Technology and Genomics

Clone

A molecule, cell, or organism that was produced from another single entity.

Restriction Enzymes

DNA-cutting enzymes (molecular scissors) that cleave the phosphodiester bond that joins adjacent nucleotides in a DNA strand.

Plasmid DNA Vectors

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

Bacteriophages

Viruses that infect cells, used in early studies to study DNA structure and replication.

Restriction Site

Specific sequences of bases where restriction enzymes bind to, recognize, and cut DNA.

Palindrome in DNA

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

4 bp and 6 bp cutters

Restriction enzymes that recognize restriction sites with a sequence of 4 or 6 nucleotides.

8 bp cutters

Restriction enzymes that have been identified to recognize restriction sites with a sequence of 8 nucleotides.

Specificity of Restriction Enzymes

Restriction enzymes show specificity for certain substrates (i.e., DNA sequences), similar to other enzymes.

Sticky Ends

DNA fragments with overhanging single-stranded ends created by some restriction enzymes.

Blunt ends

Fragments of DNA generated by cutting DNA that have double-stranded ends.

Plasmid DNA

Small circular pieces of DNA found primarily in bacteria, considered extrachromosomal DNA, approximately 1 to 4 kb in size, that can replicate independently of the chromosome.

Vectors

Pieces of DNA that can accept, carry, and replicate other pieces of DNA.

Recombinant DNA

DNA that has been formed artificially by combining constituents from different organisms.

RAC (Recombinant DNA Advisory Committee)

Formed in 1975 by the NIH to evaluate recombinant technology and establish guidelines for research.

Transformation of Bacterial Cells

A process for inserting foreign DNA into bacteria, which involves treating cells with calcium chloride, adding plasmid DNA, and applying heat.

Electroporation

A technique that applies a brief pulse of high-voltage electricity to create tiny holes in the bacterial cell wall, allowing DNA to enter.

Antibiotic selection

A method to identify recombinant bacteria by plating transformed cells on plates containing antibiotics.

Blue-white selection

A method of selecting recombinant bacteria where the presence of functional lac Z gene produces blue colonies and nonfunctional lac Z gene produces white colonies.

Insert DNA

DNA that has been cloned into a plasmid.

Human Gene Cloning

The process of cloning human genes and expressing proteins for biotechnology applications.

Insulin

The first human protein expressed via recombinant techniques.

Growth hormone

The second human protein expressed via recombinant techniques.

Functional Beta gal

Produced by the lac Z gene when it is not interrupted by an inserted gene, resulting in blue colonies.

Nonfunctional lac Z

Occurs when the lac Z gene is interrupted by an inserted gene, resulting in white colonies.

Cloning a Gene

The process of inserting a gene into a plasmid and using bacteria to replicate the recombinant DNA.

Guidelines for recombinant organisms

Published by the RAC in 1976 to ensure safe research practices.

Calcium chloride treatment

A step in the transformation process that prepares bacterial cells to take up plasmid DNA.

X gal

An artificial lactose used in blue-white selection to identify functional lac Z.

Replication of plasmid DNA

The process by which plasmid DNA is duplicated within bacterial cells.

Identification of recombinant bacteria

The goal of selection processes to distinguish transformed bacteria from nontransformed ones.

Size

Small enough to be separated from chromosomal DNA of host plasmid.

Origin of replication (ori)

Site for DNA replication that allows plasmids to replicate independently from host chromosome.

Copy number

Number of plasmids in the cell (normally small but plasmids have high-copy numbers).

Multiple cloning site (MCS)

Recognition sites for several restriction enzymes in which insert is cloned into.

Selectable marker genes

Allow to select for transformed colonies.

RNA polymerase promoter sequences

Used for transcription in vitro and in vivo.

DNA sequencing primers

Short sequences of nucleotides used to initiate DNA synthesis for sequencing.

Bacterial plasmid vectors

Can clone inserts that are smaller than 7 kb; some express eukaryotic proteins from genes poorly.

Bacteriophage vectors

Advantage: clone up to 25 kb; λ genome is linear and 49 kb.

Recombinant chromosomes

Packaged into viral particles in vitro and infect lawn of E. coli cells.

COS sites

At each end of λ are 12 bp sites that base pair together when they infect bacteria and circularize and replicate.

Obtain plaques

Zones of dead bacteria which contain millions of recombinant phage particles.

Cosmid vectors

Contain COS ends of λ DNA, plasmid ori of rep, gene for antibiotic resistance.

Bacterial Artificial Chromosomes (BAC)

Large low-copy plasmids that can accept large sizes of DNA inserts ranging from 100 to 300 kb.

Yeast Artificial Chromosomes (YAC)

Miniature version of eukaryotic chromosome containing ori or rep; two telomeres; selectable markers; centromere.

T i vector

Naturally occurring plasmids isolated from the bacterium that is a soil plant pathogen causing disease in plants.

T DNA

Codes for auxin hormone that weakens plant cell wall and infected plants divide and enlarge to form a tumor (gall).

Bacteria expression vector

Allow high-level protein expression in bacterial cells because they have a prokaryotic promoter site next to the MCS.

Problems with bacteria expression vectors

Sometimes bacteria ribosomes cannot translate eukaryote sequence or protein is not folded correctly.

DNA Libraries

Collections of cloned DNA fragments from a particular organism contained within bacteria or viruses as the host.

Genomic DNA Libraries

Libraries that contain chromosomal DNA from the tissue of interest isolated and digested with a restriction enzyme, producing many fragments that include the entire genome.

Complementary DNA Libraries (cDNA Libraries)

Libraries made from mRNA isolated from tissue, which involves synthesizing complementary DNA from mRNA using reverse transcriptase.

Whole-genome sequencing

A method that can sequence an entire genomic DNA sample without the need for inserting DNA fragments into vectors and cloning them in host cells.

Restriction Enzyme

An enzyme used to digest DNA, producing fragments for cloning in genomic libraries.

DNA Ligase

An enzyme used to ligate genomic DNA fragments and vector DNA together.

Recombinant Vectors

Vectors that contain ligated DNA fragments and are used to transform bacteria.

Introns

Non-coding pieces of DNA that are cloned in addition to exons in genomic libraries.

Exons

Coding regions of DNA that are expressed in the final mRNA product.

Double-stranded DNA

The form of DNA created from mRNA using reverse transcriptase and DNA polymerase in cDNA libraries.

Reverse Transcriptase

An enzyme that catalyzes the synthesis of complementary single-stranded DNA from mRNA.

cDNA Synthesis

The process of creating complementary DNA from mRNA, which involves reverse transcription and subsequent synthesis of the second DNA strand.

Library Screening

The process of isolating specific genes from a library using probes that are complementary to parts of the target gene.

Probes

Any DNA or RNA sequence that is complementary to some part of the target gene or sequence to be identified in a library.

Polymerase Chain Reaction (PCR)

A technique for making copies, or amplifying, a specific sequence of DNA in a short period of time.

Kary Mullis

The scientist who developed the Polymerase Chain Reaction technique in the mid-1980s.

Nucleotides

The building blocks of DNA, including dATP, dCTP, dGTP, and dTTP, used in PCR.

Buffer

A solution used in PCR to maintain the pH and ionic environment for the reaction.

DNA Polymerase

An enzyme used in PCR to synthesize new strands of DNA from the template.

Gene of Interest

A specific gene that is targeted for isolation and cloning from a library.

Cloning

The process of creating copies of a specific DNA sequence or gene.

Screening

The method used to identify and isolate specific genes from a DNA library.

Forward and Reverse Primers

Short single-stranded DNA oligonucleotides (20-30 bp long) that are complementary to nucleotides flanking opposite ends of target DNA.

Thermocycler

An instrument that takes DNA through a series of reactions called a PCR cycle.

PCR Cycle

A series of reactions consisting of three stages: Denaturation, Annealing, and Extension.

Denaturation

The first stage of a PCR cycle where the temperature is heated from 94 °C to 96 °C.

Annealing (Hybridization)

The second stage of a PCR cycle where primers hydrogen bond with complementary bases at the opposite ends of the target sequence, occurring between 55 °C and 65 °C.

Extension (Elongation)

The third stage of a PCR cycle where DNA polymerase copies target DNA from 70 °C to 75 °C.

DNA Amplification

At the end of one PCR cycle, the amount of DNA has doubled, and cycles are repeated 20-30 times.

Advantage of PCR

Can amplify millions of copies of target DNA from a small amount of starting material in a short period of time.

PCR Copy Calculation

To calculate the number of copies of target DNA starting with 1 molecule of DNA, use the equation where N represents the number of PCR cycles.

Taq DNA Polymerase

A type of DNA polymerase isolated from Thermus aquaticus, a species that thrives in hot springs.

Applications of PCR

Includes making DNA probes, studying gene expression, detecting viral and bacterial infections, diagnosing genetic conditions, and detecting trace amounts of DNA from crime scenes or fossilized tissues.

Cloning PCR Products

Is rapid and effective compared to using DNA libraries.

Disadvantage of Cloning PCR Products

Need to know something about the DNA sequence that flanks the gene of interest to design primers.

T Vector

A vector that has single-stranded thymine on each end to complementarily base pair with the adenine added by Taq polymerase on the 3′ end of all PCR products.

PCR Applications

Includes detection of DNA from fossilized dinosaur tissue.

Agarose Gel Electrophoresis

A technique to separate and visualize DNA fragments based on size.

Agarose

A substance isolated from seaweed used to create a semisolid gel for DNA separation.

Gel Percentage

The percentage of agarose used to make the gel, affecting the separation of DNA fragments; ranges from 0.5 to 2 percent.

High Percentage Gel

A gel with 2 percent agarose that allows resolution of smaller size DNA fragments.

Low Percentage Gel

A gel with 0.5 percent agarose that resolves larger size DNA fragments.

Buffer Solution

A solution that conducts electricity and is used to submerge the gel during electrophoresis.

Wells

Small depressions at the top of the gel where DNA is loaded.

Electric Current

Applied through electrodes at opposite ends of the gel to facilitate DNA migration.

Rate of Migration

Depends on the size of the DNA; smaller fragments migrate faster than larger ones.

Negatively Charged

The charge of DNA due to its sugar phosphate backbone, causing it to migrate toward the positive pole.

Migration Distance

Inversely proportional to the size of the DNA fragment; larger fragments migrate slowly.

Tracking Dye

Added to samples to monitor DNA migration during electrophoresis.

DNA Staining Dyes

Dyes like ethidium bromide that intercalate between DNA base pairs and fluoresce under ultraviolet light.