Genetic Technology

1. Genetic Technology

2. Biotechnology • Traditional vs. Modern Biotechnology • History of Biotechnology • Ethical issues

3. Genetic Engineering • Involves cutting (or cleaving) DNA from one organism into small fragments and inserting the fragments into a host organism of the same or a different species • AKA: Recombinant DNA technology • Recombinant DNA is made by connecting, or recombining, fragments of DNA from different sources

4. Transgenic Organisms • Contain recombinant DNA • Examples: Glowing tobacco (pg 349) Bt corn Golden rice

5. Steps involved in creating a transgenic organism • Step 1: Isolate the DNA fragment that will be inserted (use a restriction enzyme) • Step 2: attach the DNA fragment to the “vehicle” (virus or bacteria DNA) by “gene splicing” • Step 3: transfer the vehicle into the host organism (recombined DNA is transferred to a bacterial cell)

7. Uses of Recombinant DNA • Industry • Medicine • Transgenic animals • Agriculture • Transgenic plants

8. A Revolution in Biological Science • In mid-1970s, newly developed recombinant DNA technology provided, for the first time, powerful techniques for studying and manipulating DNA • Recombinant DNA technology allows biologists to redirect the genetic activity of organisms • Techniques and approaches include • Splicing: Specific genes can be added or removed by cutting and rearranging DNA • Genetic engineering: Modification of the DNA of an organism to produce new genes • DNA cloning: Large quantities of DNA can be obtained relatively easily by cloning cells and amplifying specific DNA sequences, both in situ (inside) and in vitro (out of) the cell • Biotechnology, the overall corporate-driven use of genetic engineering, which already has had great impact on our lives

9. History and Terminology • Biotechnology has its roots in microbiology: the study of micro- organisms (usually bacteria) • Bacteriophage (viruses of bacteria) were first used to try to understand how DNA worked (recall Hershey-Chase) • Scientists learned how to make bacteria competent for transformation (recall Griffith) by modification of the ionic environment… • Made the cell wall more permeable • Allowed the cells to take up DNA • Genetic engineering not possible prior to discovery of restriction endonucleases (restriction enzymes) by Ham Smith and Daniel Nathans (Johns Hopkins – Nobel Prize winners – 1978) • Specifically clip strands of DNA • Many different types

10. Restriction Enzymes 1 • Restriction enzymes are natural, bacterial “molecular scissors” normally used to destroy non-host (such as bacteriophage) DNA • Cut DNA at specific base pair sequences: many are palindromic • A linguistic palindrome has the same informational sequence forward and backward • MadamImadam is a linguistic palindrome • A nucleic acid palindrome has the same sequence on two antiparallel, complementary, hydrogen-bonded strands • e.g. AACGTT will pair with TTGCAA; these are palindromes • Restriction enzymes cut at the ends of the palindromic sequences (red in the example here) • They are cut in a staggered fashion:

11. New seq. L L New seq. (complementary) Restriction Enzymes 2 • Restriction enzymes snip the phosphodiester bond at a very VERY specific location based on the sequence information • Staggered cuts on ends of palindromic regions leave strands with complementary “sticky” ends when separated (usuallywith heat) • Segments of DNA with sticky ends can hydrogen bond with complementary sequences • The open spaces can be joined with purified naturally-occurring DNA ligases ( ) – process is called splicing Heat denaturation L

12. Vector DNA • A vector is a genome that carries foreign DNA into a host cell • Used to transform competent (can take up DNA) bacteria • Bacteriophage (bacterial viruses – recall Hershey-Chase) • Can carry DNA segments of up to 15kb • Engineered mammalian viruses used in mammalian cells • DNA incorporated into nuclear DNA of mammalian cell • Plasmids are small rings of double-stranded DNA that commonly occur in bacteria • Can carry DNA segments < 10kb in size 1Kb=1000 bps • Often carry genes for resistance to antibiotics • Can be used to provide a selectable marker which allows only transformed cells to live. Cells containing ampicillin resistance gene inserted by transformation can be grown on ampicillin-rich media; nontransformed cells die

13. Plasmids and Bacteria

14. Recombinant DNA Is Formedby Splicing DNA From a VectorInto Host DNA

15. PCR Is Used to Amplify DNA in Vitro • The Polymerase Chain Reaction (PCR) allows amplification of a small amount of targeted DNA in a short time. It is very simple but very powerful. • PCR has three steps: • Denaturation. A buffered mixture of primers, nucleotides, Taq polymerase and DNA fragments is heated to dissociate ds DNA into ssDNA • Annealing of primers. The solution is cooled and the primers bind to complementary sequences of the DNA at the ends • Primer Extension. DNA polymerase then uses the nucleotides to extend and make more copies of each strand • The process is repeated over and over to produce millions of copies of the original DNA strand

16. PCR

17. PCR Characteristics • DNA Taq polymerase isolated from the thermophilic bacterium Thermus aquaticus is used as it is not damaged by the heat • After 20 cycles a single fragment produces more than one million (220) copies • 30 cycles will produce a billion times the original amount (230), enough amplification to reveal the presence of a single copy of a specific target sequence • The use of PCR is virtually limitless • Criminal investigations (DNA fingerprints) from a speck of blood or single hair • Detection of genetic defects in very early embryos by collecting a few sloughed-off cells from the amniotic fluid (amniocentesis) and amplifying the DNA • Used to examine historical figures and extinct species such as mammoths and dodos • Very sensitive and samples easily contaminated

18. Gel Electrophoresis Is Widely Used to Separate DNA and RNA • DNA and RNA are negatively charged, and move through a gel at varying speeds due to different molecular lengths (sizes) • Restriction endonucleases can be used to clip the DNA • DNA fragments are loaded on a gel & an electric field is applied • Bigger DNA fragments migrate through the gel more slowly than small fragments • Fragments can be stained and visualized migrating as bands under UV light • DNA fragments can be transferred (‘blotted’) to a filter, denatured and incubated with a radioactive or fluorescent probe which will hybridize to the target sequence and be revealed by autoradiography or sensitive color digital camera

19. Southern and Northern Blotting • Blots for DNA are called Southern blots • Named after its inventor, E.M. Southern (1975) • DNA is separated on a gel • Gel is transferred onto nitrocellulose or a nylon membrane • Membrane is incubated with radioactive ssDNA probe of the gene of interest • Probe hybridizes to the blot where there is a fragment with a complementary sequence • The radioactive bands on the blot identify fragments of interest • If RNA blotted, called Northern blot

20. Western Blotting • Proteins separated in a gel • Proteins blotted onto a membrane • Antibodies specific for a particular protein are applied • Antibodies stick to target proteins ONLY • Revealed by additional antibodies attached to enzymes that precipitate a colored product

21. DNA Sequences Contain Much Information • Can determine the actual protein encoding regions… the ORFs • Regions containing transcriptional signals and RNA processing can be recognized • Amino acid sequences of proteins can be inferred from the base sequence – much faster and easier than from the protein directly • Reveals structure of chromosomes, possibly helpful in determining evolution, phylogenies, and fighting disease

22. DNA Nucleotide Sequencing • Radioactive DNA is replicated off the host template DNA • Dideoxynucleotides (ddNTPs – lacking OH at 3’ and 2’) are incorporated in small quantities in the reaction mixture to label sequences which contain those deoxybases (the ddNTPs jam DNA polymerase) • Reaction mixtures contain DNA polymerase, radioactive primers, single-stranded DNA fragment, 4 deoxynucleotides. Four tubes are prepared – each containing a different dideoxynucleotide (ddATP, ddCTP, ddGTP or ddTTP) • Fragments of varying length are formed in each mixture – the end points occur at the 4 different ddNTP • Fragments are separated based on length by electrophoresis • Autoradiography reveals the presence of the radioactively-labeled DNA fragments • The DNA sequence is literally ‘read off’ of the gel, using the 4 lanes derived from the 4 reaction tubes.

25. Restriction Fragment LengthPolymorphism Analysis • Each individual carries with it a record of the variation in genetic organization from its previous generations. There is a LOT of variation in individuals of a population • Restriction enzymes are used to cut DNA into fragments • The fragments are of different lengths in different individuals since each host DNA is unique. When three different individuals DNA are cut with a restriction endonuclease, 3 different fragment sizes are likely to be produced, unless they are identical triplets

26. An RFLP Autoradiogram • A ‘DNA fingerprint’ produced by gel electrophoresis reveals different banding patterns – restriction fragment length polymorphisms (RFLPs) • This technology is particularly important in determination of paternity and in forensics • Here, M = mother, F = father, and C = children. Note that children have all bands of M and F lanes.