MCB3020 4C: Recombinant DNA Tech and microbial genomics

biotechnology

use of living organisms to create products that are useful to humans

genetic engineering

the deliberate modification of an organism’s genome sequence

recombinant DNA technology

set of procedures used to combine genetic material from multiple sources and create DNA sequences are otherwise not found in biological organisms

cloning

generation of a larger number of identical DNA molecules from a single DNA

basic steps of cloning

1. isolate DNA to be cloned

2. use restriction enzymes to separate fragments

3. insert fragments into a cloning vector —> creates recombinant DNA

4. insert the recombinant DNA into a new host [e.g. E. coli]

5. culture host cell, growing many identical copies

restriction enzyme

endonucleases that recognize specific sites and cleave DNA

role of DNA ligase in restriction enzyme digestions

forms covalent bonds between the cloned gene and plasmid, forming recombinant DNA

reverse transcriptase

isolated enzyme from retroviruses that synthesizes double-stranded DNA from single-stranded RNA

cDNA

complementary DNA

• DNA copy of an RNA molecule

process of constructing cDNA

1. apply short poly-T primer to RNA template

2. add reverse transcriptase and the 4 nucleotides

3. add RNaseH to cut up the RNA and regenerate the RNA primers

4. add DNA polymerase and DNA ligase to synthesize the new strand

gel electrophoresis

separation of molecules according to their charge and size through a gel matrix

what charge does nucleic acid have and why?

• negative charge

because of their phosphate backbone

which electrode does nucleic acid migrate towards

the positive electrode

which sizes of DNA fragments will travel further in gel electrophoresis

shorter fragments

what dye is used on agarose gels for gel electrophoresis

ethidium bromide

what is a cloning vector

DNA molecule that can replicate independently of the host chromosome and maintain a piece of inserted foreign DNA

what are things every good cloning vector must have

• origin of replication

• selectable marker

• unique restriction sites (polylinkers or MCs)

plasmid

self-replicating, circular piece of extrachromosomal DNA

• most commonly used as a cloning vector + only found in eukaryotes

bacteriophages

a virus that infects bacteria

cosmid

plasmid vector with lambda phage cos sites that can be packaged in a phage capsid

plasmid/phage hybrid

artificial chromosomes

synthetic chromosomes that contain fragments of DNA integrated into a host chromosome

• used for cloning of large DNA fragments

genomic library

a collection of an organism’s entire genomic DNA

transformation

the uptake of naked DNA from the environment

• method of how cloning vector DNA is introduced into bacterial hosts

electroporation

use of electricity to create temporary pores in the plasma membrane —> allows for transformation

polymerase chain reaction (PCR)

a technique for quickly and easily making many copies of a very small amount of DNA

what is required for PCR

• DNA of interest

• DNA polymerase

• primers

• deoxyribonucleotide triphosphates (dNTPs)

where does dna Taq polymerase come from and why is it used in PCR

comes from thermophilic bacterium found in hydrothermal vents (Thermus aquaticus)

• able to withstand the heat of PCR

what is used to separate the the hydrogen bonds between DNA strands

high levels of heat

steps of PCR

1. Denaturation

2. Annealing

3. Extension

repeat

what happens to the DNA content of each PCR cycle

it doubles

• 6 cycles to get 64 copies aka 2⁶

heterologous gene expression

when cloned genes are expressed in the host

what methods are used to isolate or visualize the protein products of a cloned gene

polyhistidine tagging and fluorescent labeling

polyhistidine tagging (his-tagging)

adding a series of histidine amino acids residues to the N or C terminus of a protein

• facilitating the protein’s purification through affinity chromatography

fluorescence labeling

uses green fluorescent protein found in jellyfish

• when translated, cells generate strong green fluorescence

genomics

the study of the organization of genomes, the information they store, and the gene products they code for