1. An Introduction to Cloning and Recombinant DNA Chapter 13

2. 13.1 What Are Clones?  Clones Genetically identical molecules, cells, or organisms all derived from a single ancestor  Cloning The production of identical copies of molecules, cells, or organisms from a single ancestor

3.  1950s: Charles Steward grew individual carrot cells in the laboratory by using special nutrients  Cloning is used in the paper & timber industry to produce trees of uniform size, growth rate, & disease resistance

4. The 2 most common methods used in cloning animals Embryo splitting eggs are collected and fertilized in vitro embryo allowed to develop to stage of 8 to 16 cells embryo is separated into individual cells cells grow in lab & are then transplanted into surrogate mothers for development 1894 – Hans Dreisch cloned sea urchin

5. The 2 most common methods used in cloning animals Nuclear transfer / cell fusion unfertilized eggs are collected from a donor and enucleated an embryo is removed from the animal to be cloned allowed to develop to stage of 16 to 32 cells embryo is separated into individual cells and each is fused with an enucleated egg embryos are then transplanted into surrogate mothers for development 1986 –cloned sheep (NOT Dolly!) This technique is more difficult but can result in a larger number of offspring

6. Why ~16 cell embryos?  The genetic information in some cells is slightly modified as embryo develops (the cells may become specialized / differentiated) How is Dolly different?  In 1997 a differentiated cell from the udder of an adult sheep was used to produce a clone animal - Dolly

7. Cloning Animals: Pro & Con PRO  Produce herds of identical animals with superior wool, milk, or meat production CON  Loss of diversity  Susceptibility to disease, pests, or changing environmental conditions

8. Cloning of DNA / Genes  Methods for cloning DNA are referred to as recombinant DNA technology  DNA clones are used to find genes, map them, and transfer them between species  Cloning technology is used to find carriers of genetic disorders, perform gene therapy, and create disease-resistant plants

9. What’s Needed to Clone DNA?  A way to cut DNA at specific sites  Restriction enzymes (produced by bacteria to protect against viral infections)  A carrier molecule to hold DNA for cloning  Vector is usually a bacterial plasmid (a circular DNA molecule that is self-replicating)  A place where the DNA can be copied (cloned)  Host cell – most common is the bacterium E. coli

10. Why is a vector necessary? Vectors are needed because linear DNA (such as human DNA) cannot be replicated within a host cell and passed on to progeny. Vectors are self-replicating and can carry the DNA into the host cells for replication

11. 13.2 Cloning Genes Is a Multistep Process See page 294, 295  Cut DNA at specific sites  Connect the fragments to vectors using DNA ligase, creating recombinant DNA molecules  Transfer the recombinant DNA molecule to a host cell (where it is copied to form clones)  Retrieve the cloned DNA fragment from the vector in large quantities  Turn on gene & collect & purify gene product

12. Recognition & cutting sequences … Are palindromes that read the same on either DNA strand when read for 5’ to 3’ direction This ensures that the sequence will be recognized no matter which end of the molecule the sequence is approached from

13. Cut, Connect, & Introduce  Cut at recognition sites using restriction enzymes  Connect to vector using DNA ligase  Vector introduced or transferred to vector

14. Different restriction enzymes have different recognition sequences

15. Identifying Bacteria Colonies With Recombinant DNA  Not all vectors successfully incorporate the recombinant DNA so scientists have to have a way to identify the bacteria colonies with recombinant DNA.  Usually, the DNA is inserted in a region that codes for antibiotic resistance;  if the DNA insertion is successful then the resistance gene is nonfunctional and the colonies with human DNA inserted will not grow on growth plates that have the antibiotic as part of the growth medium

16. Colonies not resistant can grow on one plate but not on the plate loaded with the antibiotic

17. Now its time to score! Now that the host cell has the recombinant DNA the “goal” is ready to be scored!  The purpose of the clones is to produce a “library” of specific gene sequences for researches to use  See slide 8

18. 13.3 Cloned Libraries  Libraries are resources for gene studies  To make a human genomic library using bacterial plasmids ~8 million plasmid clones would be needed (plasmids generally can carry DNA fragments of about 1700 base pairs long)  YACs (yeast artificial chromosomes) are vectors that use eukaryotic yeasts as host cells; YACs can carry DNA fragments of 1 million base pairs. A human genome library could be carried in just over 3000 clones  Search through 8 million or search through 3000?

19. Genetics in Society Asilomar: Scientists Get Involved  An international conference was held at Asilomar, California, to consider the possible dangers of recombinant DNA technology In 1976, guidelines were set in place for experiments using recombinant bacteria New guidelines were published in 1982 No experiments are currently prohibited

20. 13.4 Finding a Specific Clone in a Library  Clones for specific genes can be recovered from a library by using probes to screen the library  Probe A labeled nucleic acid used to identify a complementary region in a clone or genome

21. Could there be an easier way?  YES!  A technique invented in 1986 called polymerase chain reaction (PCR).

22. 13.5 A Revolution in Cloning: The Polymerase Chain Reaction  Polymerase chain reaction (PCR) A method for amplifying DNA segments using cycles of denaturation, annealing to primers, and DNA polymerase-directed DNA synthesis  PCR copies a DNA molecule without restriction enzymes, vectors, or host cells Faster and easier than conventional cloning

23. First Step in PCR: Denaturation 1. DNA is heated to break the hydrogen bonds between the two polynucleotide strands Two single-stranded DNA molecules serve as templates

24. Second Step in PCR: Annealing 2. Short nucleotide sequences (primers for DNA replication) are mixed with the DNA and bind to complementary regions on single-stranded DNA Takes place at lower temperature Primers are 20-30 nucleotides long, synthesized in the laboratory

25. Third Step in PCR: DNA Synthesis 3. The enzyme Taq polymerase is added to synthesize a complementary DNA strand Taq is a DNA polymerase from a bacterium found in hot springs  These three steps make up one PCR cycle

26. Keep In Mind  The polymerase chain reaction (PCR) copies DNA without cloning

27. 13.6 Analyzing Cloned Sequences  Cloned sequences are characterized in several ways, including Southern blotting and DNA sequencing  Southern blot A method for transferring DNA fragments from a gel to a membrane filter, developed by Edwin Southern for use in hybridization experiments

28. Southern Blotting Can Be Used to Analyze Cloned Sequences  Southern blotting uses gel electrophoresis to separate DNA fragments DNA fragments migrate through the gel from a negative pole to a positive pole Small fragments move faster than large fragments  Radioactive probes identify blotted bands

29. DNA Sequencing Can Be Done for an Entire Genome  DNA sequencing A technique for determining the nucleotide sequence of a fragment of DNA Basic method used in genome projects  There are several ways to sequence DNA

30. Automated Sanger Method  DNA is separated into strands  DNA polymerase, a primer, and four kinds of altered nucleotides are added Each nucleotide fluoresces a different color  When an altered nucleotide is added, synthesis stops; strands of every length accumulate Fragments are separated by length and scanned with a laser that reveals the fluorescent tag

31. Automated DNA sequencing

32. Genetic Journeys: DNA Sequencing  In 1977, Fred Sanger sequenced the 5,400 nucleotides in the genome of a virus  Automated methods allowed the human genome (3.2 billion nucleotides) to be sequenced  DNA sequencing is one of the basic methods in recombinant DNA technology

33. Chapter 13 Assignment  Questions 1 – 18, 21  #6 I’ll give hint