Bio 309 Chapter 13 Recombinant DNA Technology

Bio 309 Chapter 13 Recombinant DNA Technology

1. Cloning Concepts

A. What are clones?

B. Cloning plants from single cells

C. Several methods for cloning animals

1. splitting embryos

2. nuclear transfer

3. stem cell technology

a. procedures

1. G0 arrested donor cell

2. remove nucleus from donor cell

3. remove nucleus from recipient egg

4. fuse egg with donor G0-arrested nucleus

5. transfer to surrogate

b. types of stem cell cloning

1. reproductive cloning

2. therapeutic cloning

c. stem cell clones are not exact replicas

1. telomeres of chromosomes of donor nuclei are shorter

2. genomic imprinting when chromosomes are passed via germline

3. somatic cells accumulate mutations

4. X-inactivation pattern

5. mitochondria is from recipient cell not donor cell

6. coat color differences-pigmented cells move about differently

7. environmental factors during embryonic/fetal development

1. nutrition, stress, exposure to environmental diseases

d. why does stem cell cloning fail?

1. Meiosis not involved (meiosis in the female completes at fertilization)

2. diploid nucleus is plunked into oocyte cytoplasm where signals direct it do what a female secondary oocyte tends to do

a. shed half of chromosomes as polar body--haploid-lethal

b. replicates DNA--tetraploid--lethal

e. bioethical issues of stem cell cloning

1. violation of rights of early-stage embryos versus violation of rights of individuals who might benefit from such therapy

2. 2001 USA legislation outlaw creating or selling "any embryo produced by human cloning for reproductive or therapeutic purposes"

3. failure to remove "cloning for therapeutic purposes" results in

a. private funding cannot be used for cloning-therapeutic purposes

b. U.S. scientists departing for other countries, England and Asia

D. Steps in process of cloning DNA

1. isolate plasmid DNA and human DNA

2. cut both DNA with the same endonuclease

3. combine both DNA's--cut ends re-associate or anneal

4. seal gaps with DNA ligase

5. creates recombinant DNA molecules composed of human DNA and vector DNA

6. transfer vector into bacteria

7. at each bacterial cell division, the plasmid is replicated resulting in many copies or clones of the DNA insert

8. lyse bacteria--extract recombinant plasmids

9. cloned human DNA can be released from plasmid with same restriction enzyme used for cloning

10. cloned DNA can be used directly, put in different vectors, such as expression vector and transfected into cells

Chapter 12, continued

E.. cDNA libraries

F. Revolution in cloning: Polymerase Chain Reaction (PCR)--1986

a. Reverse transcriptase (RT) RNA---DNA

b. Polymerase Chain Reaction (PCR) (PCR cycle)

1. DNA amplified by heating to break hydrogen bonds, yielding single stranded DNA

2. Short nucleotide sequences act as primers for DNA replication added

3. Enzyme, DNA polymerase, begins at primers and synthesizes a DNA strand complementary to the region between the primers, a process called "primer extension"

4. Example: 10 cycles = 1,024 copies, 30 cycles = 1,073,741,820 copies

2. Tools or procedures used in molecular genetics

A. Southern blotting

B. Northern blotting

C. Western blotting