BIO 325 Genetics Recombinant DNA Technology Be of good cheer Do not think of today s failures but of the success that may come tomorrow You have set yourselves a difficult task but you will succeed if you persevere and you will find a joy in overcoming obstacles Remember no effort that we make to attain something beautiful is ever lost Helen Keller Address to the American Association to Promote the Teaching of Speech to the Deaf at Mt Airy Philadelphia Pennsylvania 8 July 1896 Note Helen Keller entered Radcliffe College in the fall of 1900 and received her bachelor of arts degree cum laude in 1904 Recombinant DNA Technology Recombinant DNA Technology Recombinant DNA technology involves the use of in vitro molecular techniques to isolate and manipulate DNA fragments Recombinant DNA Technology Recombinant DNA technology involves the use of in vitro molecular techniques to isolate and manipulate DNA fragments What are recombinant DNA molecules Recombinant DNA Technology Recombinant DNA technology involves the use of in vitro molecular techniques to isolate and manipulate DNA fragments What are recombinant DNA molecules What has this technology enabled researchers to study Recombinant DNA Technology Recombinant DNA technology involves the use of in vitro molecular techniques to isolate and manipulate DNA fragments What are recombinant DNA molecules What has this technology enabled researchers to study What are some of the applications of these technologies Gene Cloning Molecular biologists frequently focus on the structure and function of proteins or the genes that encode them Gene Cloning Molecular biologists frequently focus on the structure and function of proteins or the genes that encode them What is gene cloning and what are some of the uses of gene cloning Gene Cloning Cloning experiments may involve two kinds of DNA molecules chromosomal DNA and vector DNA Gene Cloning Cloning experiments may involve two kinds of DNA molecules chromosomal DNA and vector DNA What is a vector and what does it do Gene Cloning Cloning experiments may involve two kinds of DNA molecules chromosomal DNA and vector DNA What is a vector and what does it do What is a host cell and what does it do Gene Cloning What are plasmids Gene Cloning Plasmids that contain genes that confer resistance to toxic compounds and antibiotics are called R factors Gene Cloning Plasmids that contain genes that confer resistance to toxic compounds and antibiotics are called R factors What s the deal with a plasmids origin of replication Gene Cloning Plasmids that contain genes that confer resistance to toxic compounds and antibiotics are called R factors What s the deal with a plasmids origin of replication What s a selectable marker Gene Cloning A viral vector is a virus that contains a chromosomal gene Gene Cloning A viral vector is a virus that contains a chromosomal gene Other vectors such as cosmids BACs and YACs are described later in the course Gene Cloning Enzymes called restriction endonucleases or restriction enzymes are used to cut DNA They recognize palindromic sequences Gene Cloning Enzymes called restriction endonucleases or restriction enzymes are used to cut DNA They recognize palindromic sequences What s a palindromic sequence Gene Cloning Enzymes called restriction endonucleases or restriction enzymes are used to cut DNA They recognize palindromic sequences What s a palindromic sequence Let s see how all of this works What are sticky ends and what does this mean to recombinant DNA technology Do you see what s happening Inserting a Piece of DNA into Another DNA G A A T T C G A A T T C C T T A A G C T T A A G G A A T T C C T T A A G G A A T T C G A A T T C C T T A A G C T T A A G G A A T T C C T T A A G Cut with a particular restriction enzyme G A A T T C G A A T T C C T T A A G C T T A A G G A A T T C C T T A A G G A A T T C G A A T T C C T T A A G C T T A A G G C T T A A A A T T C G G A A T T C G C T T A A C T T A A G G A A T T C Cut with a the same restriction enzyme G C T T A A A A T T C G G A A T T C G C T T A A C T T A A G G C T T A A G A A T T C A A T T C G G A A T T C C T T A A G G C T T A A G A A T T C C T T A A G A A T T C G G A A T T C C T T A A G A A T T C G G C T T A A G C T T A A A A T T C G G A A T T C C T T A A G A A T T C G G C T T A A G C T T A A A A T T C G G A A T T C C T T A A G G A A T T C C T T A A G G A A T T C C T T A A G G A A T T C C T T A A G G A A T T C G A A T T C C T T A A G C T T A A G Ligate Gene Cloning Gene cloning involves the insertion of DNA fragments into vectors which are then propagated within host cells Gene Cloning Gene cloning involves the insertion of DNA fragments into vectors which are then propagated within host cells Let s take a closer look Gene Cloning Host cells carrying recombinant vectors may then be isolated Gene Cloning Host cells carrying recombinant vectors may then be isolated Then what do you need to figure out Gene Cloning cDNA can be made from mRNA via reverse transcription Gene Cloning cDNA can be made from mRNA via reverse transcription Let s take a closer look Gene Cloning The DNA made in this manner is called complementary DNA or cDNA Gene Cloning The DNA made in this manner is called complementary DNA or cDNA cDNA lacks introns This makes it simpler to insert cDNA into vectors for cloning Polymerase Chain Reaction Polymerase Chain Reaction Another way to copy DNA without the aid of vectors and host cells is a technique called polymerase chain reaction Chromosomal DNA Gene o f interes t Primer binding near one end of the gene A different primer binding near the other end of the gene Many PCR cycles Many copies of the gene of interest flanked by the …