Genetic engineering basics

Genetic engineering involves the manipulation of naturally occurring processes and enzymes.











Recombinant DNA (rDNA): DNA made by joining pieces from two or more different sources


Genetic engineering:

• extraction/syntheses a gene(s) from one organism
• transfer gene(s) into another organism (of same or different species)
• --> gene is expressed in new host

Plasmids as vectors in gene cloning
Plasmids are small, circular pieces of double-stranded DNA

• small --> easy to use
• exist naturally in bacteria --> bacteria take up plasmids from surroundings
• can be produced artificially 
• double stranded: can insert genes from prokaryotes and eukaryotes
• replicate independently in bacteria
• can be transferred between different bacterial species

Promoters - control expression of genes --> ensure high levels of gene expression

Promoter binds to DNA strand

• allows RNA polymerase to bind to DNA
• ensures RNA polymerase recognizes which is the template strand

---> promoter region = transcription startpoint

Enzymes:

Restriction endonuclease: restrict viral infections by recognising and breaking down DNA of invading viruses.

---> binds to specific target site on DNA (sequence of bases) --> cut sugar-phosphate backbone:

• straight across = blunt ends
• staggered fashion = sticky ends*

* sticky ends: short lengths of unpaired bases; easily form hydrogen bonds with complementary base sequences on other fragments of DNA cut with the same restriction enzyme

Reverse transcriptase: 

• uses single-stranded mRNA as the template
• reverses the transcription process: produces single stranded DNA
• single-stranded DNA + DNA polymerase = double-straned complementary DNA (cDNA)

DNA ligase: links together sugar-phosphate backbones of DNA molecules and plasmid ---> produces a closed circle of double-stranded DNA containing the new gene = "recombinant DNA"

19.1 Principles of genetic technology  Genetic engineering involves the manipulation of naturally occurring processes and enzymes.  Genome sequencing gives information about the location of genes and provides evidence for the evolutionary links between organisms. a) define the term recombinant DNA  b) explain that genetic engineering involves the extraction of genes from one organism, or the synthesis of genes, in order to place them in another organism (of the same or another species) such that the receiving organism expresses the gene product  c) describe the principles of the polymerase chain reaction (PCR) to clone and amplify DNA (the role of Taq polymerase should be emphasised)  d) describe and explain how gel electrophoresis is used to analyse proteins and nucleic acids, and to distinguish between the alleles of a gene (limited to the separation of polypeptides and the separation of DNA fragments cut with restriction endonucleases)  e) describe the properties of plasmids that allow them to be used in gene cloning  f) explain why promoters and other control sequences may have to be transferred as well as the desired gene  g) explain the use of genes for fluorescent or easily stained substances as markers in gene technology  h) explain the roles of restriction endonucleases, reverse transcriptase and ligases in genetic engineering  i) explain, in outline, how microarrays are used in the analysis of genomes and in detecting mRNA in studies of gene expression