Lecture 13: DNA Technology. DNA Sequencing. DNA Sequencing Genetic Markers - RFLPs polymerase chain reaction (PCR) products of biotechnology

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1 Lecture 13: DNA Technology DNA Sequencing Genetic Markers - RFLPs polymerase chain reaction (PCR) products of biotechnology DNA Sequencing determine order of nucleotides in a strand of DNA > bases = A, T, G, C using an automated sequencer ingredients > DNA template - many copies > nucleotides for DNA synthesis > fluorescent termination nucleotides (stops DNA synthesis) > DNA primers: short pieces (<20 nt) that match ends of the template > DNA polymerase - catalyze DNA synthesis 1

2 DNA Sequencing continued DNA Sequencing - the movie (Celera Genomics) (www.celera.com/geneed/c/c/celera_demo/index.html) 1. primers attach to template 2. polymerase starts DNA elongation by adding nucleotides 3. stops randomly when termination nucleotide is used DNA copies of varying lengths with known ends (diff color) 4. construct sequence by reading termination nucleotide from shorted to longest fragment done by automated sequencers DNA Sequencing 2

3 Genetic Markers DNA varies from individual to individual (twins excepted) > differences in DNA sequences > one or more nucleotides may differ importance of these markers > unique identity ( genetic finger prints ) > indicate genetic diseases how do you detect these differences? > sequence genome directly > human genome project = ~3 billion base per bp + 10 yr > look for indirect evidence of genetic differences? restriction fragment length polymorphism (RFLP) > +/- of specific DNA sequences varying lengths RFLPs type of genetic analysis > looks for presence or absence of specific sequences of DNA overview > get DNA from patient or suspect > cut up DNA using restriction enzymes > results in DNA fragments of varying lengths > test for presence or absence of specific DNA fragments > see fig 12.11C (p241) 3

4 Steps in RFLP Analysis DNA extraction > break up cells - physical and chemical means > isolate DNA cut up DNA using restriction enzymes > enzymes cut DNA at specific sites > many different enzymes available > DNA in smaller fragments RFLP Analysis continued gel electrophoresis > separate DNA according to size and charge > DNA negatively charged > place DNA at one end of a gel (jello-like substance) encased in glass > hook up gel to an outlet > DNA move to positive end / small fragments move faster 4

5 RFLP Analysis continued Blotting > separate the two strands of DNA (chemical process) > transfer DNA from gel to filter paper Probing > single stranded DNA > have radioactive phosphorous > known sequence > pour over DNA stuck on filter paper > probe sticks to DNA of interest RFLP Analysis continued detect genetic markers > overlay the filter paper with photographic film > radiation from probe forms image on film > develop film to visualize genetic markers (radioactive bands) filter paper photo film 5

6 Overview of RFLP Analysis cut up DNA > restriction enzyme separate DNA fragments > gel electrophoresis - small fragments travel further on gel > DNA occur as bands on gel blot DNA onto filter paper > add chemicals to split DNA into single strands > overlay gel with filter paper d probe for specific DNA > radioactive DNA of known sequence > bind to specific sequences of DNA (genetic markers) visualize genetic markers > overlay filter paper with photographic film and expose > develop to see presence of genetic markers RFLPs - Genetic Screening identify genetic diseases before symptom are detected > some diseases are due to mutations > e.g., hemophilia, PKU, cystic fibrosis > mutation will affect RFLP pattern > genetic screening consists of examining RFLPs > first step in locating the disease gene 6

7 RFLPs - Forensic Science match DNA samples from crime scenes with DNA from potential suspects RFLP s can be unique to individuals > genetic fingerprinting only small sample of cells are needed > drop of blood > skin under finger nails what is the probability that two people (non-twins) will have same RFLP patterns? > depends on the number of genetic markers compared > can be lower than 1 in a billion! not enough DNA? Not Enough DNA? Polymerase Chain Reaction - PCR replicate conditions for making copies of DNA outside the cell copying occur in cycles > separate strands > make copies of each strand number of copies double each cycle > after 5 cycles = 32 > after 10 cycles = 1,024 > after 15 cycles = 32,768 > after 20 cycles = 1,048,576 > after 25 cycles = 33,444,432 > after 30 cycles = 1,073,741,824 7

8 Products of Biotechnology some products of biotechnology > gene therapy > genetically modified organisms both are recombinant DNA technologies > DNA of more than one organism requires use of a vector > a way of inserting genes into the target cells > viruses (retroviruses) - made harmless > plasmid - circular pieces of DNA that can general steps > isolate useful gene > insert into vector > allow vector to insert DNA into host cells > all host cells to develop into adult Gene Therapy treatment of genetic diseases > replace defective gene not yet fully developed > not a one time fix > treatment must be continual example > defective gene not producing a protein in bone cells > treat patient with recombinant bone cells uses retrovirus as vector > restroviruses insert DNA in the host > made harmless -- does not kill host cells 8

9 Genetically Modified Organisms (GMOs) plasmids used to insert useful genes into target organisms e.g., StarLink corn pesticide gene (bt) into corn plants > bt gene isolated from a bacterium > bt corn can be used for feed > not clear if bt gene is harmful to humans Ethics of Genetic Engineering is there a difference between breeding species in the field and genetic engineering in the lab? 9

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