20.1 Recombinant DNA Technology

• Recombinant DNA technology is a set of methods that allows researchers to mix and match (“recombine”) specific DNA sequences from any organism to create DNA molecules not found in nature.

• Biotechnology is the engineering of genes, cells, and organisms for basic research as well as practical purposes

• One of the most basic requirements of recombinant DNA technology is the ability to produce many copies of a gene or other DNA sequence of interest, a process referred to as DNA cloning.

• Researchers clone a sequence of DNA by inserting it into a small, circular DNA molecule called a plasmid.

• When a plasmid is used to make copies of a foreign DNA sequence, it is called a cloning vector, or simply a vector.

• A restriction endonuclease cuts DNA molecules at specific base sequences called recognition sites.

• With staggered cuts, the resulting DNA fragments are described as having sticky ends, because the single-stranded bases exposed on the plasmid are complementary to the single stranded bases exposed on the foreign DNA.

• DNA ligase is the he enzyme that connects Okazaki fragments during DNA replication

• Cells that take up DNA from the environment and incorporate it into their genomes are said to undergo transformation.

• DNA taken directly from an organism is called genomic DNA

• DNA that is transcribed from RNA is called complementary DNA, or cDNA.

• Another important use of cDNAs is in deducing the amino acid sequence of the polypeptide encoded by a gene.

20.2 The Polymerase Chain Reaction

• Polymerase chain reaction (PCR) provided a quicker way to clone DNAs.

• DNA fingerprinting (also known as DNA profiling or DNA typing) refers to any technique for identifying individuals based on the unique features of their genomes.

• The type of tandem repeats used in most current DNA fingerprinting approaches is short tandem repeats (STRs) (also known as microsatellites or simple sequence repeats [SSRs]).

20.3 Analyzing Genomes

• The first method for sequencing DNA that came into widespread use is called dideoxy sequencing.

• In shotgun sequencing, many copies of a genome are broken up randomly into a set of fragments of various sizes.

• The first high-quality genome sequence obtained of a given species is called a reference genome.

• Bioinformatics is a field that fuses mathematics, computer science, and biology to manage and analyze sequence data.

• Genomics is the branch of biology that obtains and analyzes genome sequences to gain insights into life.

• Once this massive sequence data set is collected, the first task is to identify which regions constitute genes and other functionally important sequences which is called genome annotation.

• The presence of a long stretch of codons without a stop codon which is an open reading frame, or ORF is a good indication of a protein-coding sequence.

• If genes are homologous, it means they are similar (show homology) because they are related by descent from a common ancestor.

• The idea is to look for associations (co-inheritance) between a phenotype of interest, such as adult-onset diabetes, and DNA sequences at known locations in the genome, called genetic markers.

• Single nucleotide polymorphism (SNP) is a site in DNA that varies at a single base pair.

• An animal model is a laboratory animal with disease symptoms that parallel those of a human disease.

20.4 Insights into Genomes

• Metagenomics is the only way to get a good handle on types of microbes present in an environment.

• Transposable elements are DNA segments that can move from one place to another within the genome.

• Within a species, genes that are similar to each other in structure and function are considered to be part of a gene family and to have arisen from a shared ancestral sequence 1rough gene duplication.

• A common way that genes are duplicated is through a process called unequal crossing over during meiosis.

• Although mutations in duplicated regions can create genes with new functions, more often, mutations lead to nonfunctional genes, called pseudogenes.

20.5 Genome Editing

• The CRISPR locus is transcribed into a long pre-crRNA

• A crRNA binds to the complementary DNA sequence of an invading virus, but it doesn't act alone.

• Cas proteins are enzymes (more precisely, endonucleases) that serve to cut DNA that is complementary to a crRNA.

20.6 Gene Therapy

• Gene therapy is any approach to treating or curing disease that involves modifications of the genome.

• In order for gene therapy to be successful, the requirements include:

  • The disease must be due to defects in a single gene

  • The sequence of the wild-type allele must be known

  • There must be a way to introduce this therapeutic allele into affected individuals and have it expressed in the correct tissues, in the correct amount, and at the correct time.

• These modified viruses are referred to as vectors, and their genomes have been altered to allow the incorporation of therapeutic genes and to disable replication of the virus in target cells.

20.7 New Frontiers: Functional Genomics, Proteomics, Systems and Synthetic Biology

• One of these uses a DNA microarray to assess the expression of thousands of genes at a time.

• Researchers use microarrays in many ways, such as to establish which genes are transcribed in different organs and tissues, in cancers, or in response to signals.

• The most common approach for discover“ ing genome-wide patterns of gene expression is a method called deep sequencing or RNA-seq

• Biologists use the term transcriptome in referring to the complete set of RNA molecules that have been transcribed in a particular cell, and proteome in referring to the complete set of proteins that are produced.

• It follows that proteomics is the large-scale study of all the proteins in a cell or organism.

• Systems biology seeks knowledge of how the network of interactions between the individual parts of a biological system lead to properties of life.

• Emergent properties are properties that arise at one level of organization from the interaction of simpler elements at a lower organizational level.

• Synthetic biology pursues knowledge by building biological systems and discovering what properties they demonstrate.