DNA Tools & Biotechnology Chapter 20

YOU MUST KNOW The terminology of biotechnology How plasmids are used in bacterial transformation to clone genes The key ideas that make PCT possible and applications of this technology How gel electrophoresis can be used to separate DNA fragments or protein molecules Information that can be determined from DNA gel results, such as fragment sizes and RFLP analysis

Biotech Terms -Genetic Engineering – process of manipulation genes and genomes -Biotechnology – the process of manipulating organisms or their components for the purpose of making useful products -Recombinant DNA – DNA that has been made artificially using DNA from other sources or species

Gene cloning -process by which scientists can produce multiple copies of specific segments of DNA that they can then work with in the lab

Restriction enzymes -Cut DNA at specific sites (restriction sites) – usually palindromes -Found naturally in bacteria – used to cut up invading viral DNA -Once DNA is cut, results in restriction fragments with sticky ends

Steps of Gene Cloning Gene of interest identified and isolated -Also need a cloning vector which will carry the gene – most often a bacterial plasmid

2. Gene of interest and vector are cut with the same restriction enzymes, resulting in complimentary sticky ends

3. The target DNA is fused into the plasmid using DNA ligase 4. The vector carrying the gene of interest is introduced into bacterial cells by transformation

5. Select for cells that have been transformed – usually gene of interest is linked to gene for antibiotic resistance and then the bacteria is grown on plates containing antibiotics – only the cells that have taken up the new plasmid grow

What can you do with cloned genes? -Nucleic acid hybridization – if the sequence of the gene is known, a probe can be made to track the gene -Probes – short sequence of bases that are complimentary to part of the gene and are radioactive or fluorescent

-PCR – polymerase chain reaction -amplifies (makes lots of copies) a particular piece of DNA -allows us to do other techniques with a small sample of DNA

-Gel electrophoresis – used to separate fragments of DNA or proteins -Restriction Fragment Length Polymorphisms (RFLPs) -pieces of DNA created when DNA is cut up by restriction enzymes -create specific banding patterns that can be used to identify individuals and diagnose disease ***DNA is stained so it can be seen in the gel

Genomic library – set of thousands of recombinant plasmid clones which each contain a piece of the original genome being studied

cDNA – complementary DNA – DNA created in a lab by using mRNA and reverse transcriptase -contains only exons

DNA microarray assays 1. Small amounts of single-stranded DNA (ssDNA) fragments (each a gene) are fixed to a glass slide in a grid – called a DNA chip 2. mRNA from cells being tested are isolated and used to make cDNA and tagged with fluorescent dye 3. cDNA binds to ssDNA on chip, indicating which genes are turned on in the cell

Cloning

Therapeutic cloning -Production of stem cells – cells that are undifferentiated -embryonic stem cells – pluripotent – capable of differentiating into many different types of cells -multipotent stem cells – more differentiated than embryonic, but still capable of becoming several different types of cells

Practical Applications -Diagnosis of disease – use RFLP analysis and amplification -Gene therapy – alteration of an afflicted individuals genes http://learn.genetics.utah.edu/content/genetherapy/gtsuccess/ -Production of pharmaceuticals – human insulin and growth hormone

-Transgenic animals – eggs fertilized in vitro and desired gene is inserted into the nucleus of the embryo

-Forensic Science - DNA samples from crime scenes can be compared to known samples (genetic profiles) or used in paternity testing -Environmental cleanup – engineer microbes that can break down harmful substances such as heavy metals from mining -Agriculture – GMOs - insert genes into crops the increase productivity and efficiency