BIOL. 1406 Chapter 20 DNA Tools and Biotechnology

DNA Sequencing, DNA Cloning, PCR Amplification, expressing genes, analyzing gene expression, and gene editing

Technology for DNA Sequencing and Manipulation

the complementarity of two DNA strands is the basis for it; the base pairing of one strand is complementary to the sequence of another

Direct Manipulation of DNA for practical purposes

a process by which a gene’s complete nucleotide sequence is determined; can be done by dideoxy/ chain termination sequencing, sequencing by synthesis, or third-generation sequencing

an automated procedure that determines DNA sequencing, developed by Frederick Sanger

many DNA fragments are copied to produce an enormous number of identical fragments; a single strand of each fragment is immobilized and the complementary strand synthesized one nucleotide at a time; thousands or hundreds of thousands of fragments about 300 nucleotides long can be sequenced in parallel; “high-throughput” technology

Techniques are faster and less expensive; in this method, a single DNA molecule is sequenced as it moves through a pore (nanopore) in a membrane; n one approach, each base is identified by the way it interrupts an electric current as it passes through the pore; associated software allows identification and analysis of the DNA sequence

to work directly with specific genes, scientists prepare well-defined DNA segments in multiple copies by this process

small, circular DNA segments that replicated independently of bacterial chromosomes; reproduction of plasmid with recombinant DNA results in cloning of the molecule with the foreign DNA

insertion of a DNA molecule into a plasmid that, in result, contains DNA from two different sources

production of multiple copies of a gene; a type of DNA cloning; useful amplifying of genes to produce a protein product for research, medical, and other purposes

a plasmid used to replicate foreign DNA

readily obtained, easily manipulated, easily introduced into bacterial cells, rapidly multiplied once in bacteria

Proteins that cut DNA molecules at specific DNA sequences called restriction sites, yielding restriction fragments; the most useful enzyme cuts the DNA in a staggered way, producing fragments wit at least one sticky end.

can bond with complementary sticky ends of other fragments

an enzyme that seals the bonds between restriction fragments, allowing researchers to join DNA fragments from different sources

technique that uses a gel made of polymer that has microscopic holes of different sizes, through which shorter fragments can travel; used to separate and visualize fragments

a reaction that amplifies DNA, making many copies of specific target sequence of DNA; involves a three-step cycle: 1) heating (denaturing), 2) cooling (annealing), 3) extension; the cycle produces an exponentially growing population of identical DNA molecules; the process uses primers (short single-stranded DNA molecules complementary to sequences on either side of target sequence; key to reaction is a heat-stable DNA polymerase called Taq polymerase

a heat-stable DNA polymerase used in PCR

a DNA polymerase that may be used in PCR

can be designed to include restriction sites that allow the product to be cloned into a plasmid vector

A cloning vector that is employed by scientists that contains a highly active bacterial promoter

1) different DNA control systems (including promoters) 2) presence of introns a cloning vector with a highly active bacterial promoter is implemented; a cDNA is used (introns already cut out)