What is Gene Technology?

1. What is Gene Technology? • Gene technology is a broad field which includes analysis of DNA as well as genetic engineering and other forms of genetic modification. • Gene technologies have great potential to benefit humanity through: • increasing crop production • increasing livestock production • preventing and fighting disease • reducing pollution and waste • producing new products • detecting and preventing crime

2. TOOLS and TECHNIQUES

3. Restriction Enzymes Recognition Site cut Recognition Site The restriction enzyme EcoRI cuts here cut cut • Restrictionenzymes are used as “molecularscalpels / scissors” , to cut up DNA. • Restrictionenzymes cut DNA molecules at very precise sequences of 4 to 8 base pairs called recognition sites. GAATTC GAATTC DNA CTTAAG CTTAAG

4. What’s in a name? • Restriction enzymes are named after the micro organism from which they are isolated • e.g. BamHI was isolated from the bacteria Bacillus amyloliquefaciens strain H.

5. Sticky Ends A restriction enzyme cuts the double-stranded DNA molecule at its specific recognition site A A T T C G C T T A A G The cuts produce a DNA fragment with two “sticky” ends A A T T C A A T T C G G The two different fragments cut by the same restriction enzyme have identical sticky ends and are able to join together G G C T T A A C T T A A When two fragments of DNA cut by the same restriction enzyme come together, they can join by base-pairing Restriction enzyme: EcoRI • It is possible to use restriction enzymes that cut leaving an overhang; a so-called “sticky end”. • DNA cut in such a way produces ends which may only be joined to othersticky ends with a complementary base sequence. Fragment A A T T C G C T T A A G Restriction enzyme: EcoRI Sticky end DNA from another source

6. It is possible to use restriction enzymes that cut leaving no overhang; a so-called “blunt end”. Blunt Ends C C C C C C G G G G G G G G G C C C G G G G G G C C C C C C DNA G G G C C C C C C G G G G G G C C C C C C G G G G G G C C C cut cut C C C G G G C C C G G G Recognition Site Recognition Site Restriction enzyme cuts here The cut by this type of restriction enzyme leaves no overhang DNA from another source A special group of enzymes can join the pieces together

7. Ligation DNA fragments produced using restriction enzymes may be reassembled by a process called ligation. Pieces of DNA are joined together using an enzyme called DNA ligase. DNA of different origins produced in this way is called recombinant DNA because it is DNA that has been recombined from different sources. Two pieces of DNA are cut using the same restriction enzyme. The two different DNA fragments are attracted to each other by weak hydrogen bonds. This other end of the foreign DNA is attracted to the remaining sticky end of the plasmid. A A T T C G Plasmid DNA fragment Foreign DNA fragment G C T T A A

8. Annealing Detail of Restriction Site Restriction sites on the fragments are attracted by base pairing only Gap in DNA molecule’s ‘backbone’ A T T A C G T A A T G C Plasmid DNA fragment Foreign DNA fragment C G T A A T G C T A A T When the two matching “sticky ends” come together, they join by base pairing. This process is called annealing.

9. Recombinant DNA Plasmid Detail of Restriction Site A T T A C G A T A T G C Recombinant Plasmid DNA Fragments linked permanently by DNA ligase No break in DNA molecule C G T A A T G C T A A T • The fragments of DNA are joined together by the enzyme DNA ligase, producing a molecule of recombinant DNA. • These combined techniques of using restriction enzymes and ligation are the basic tools of geneticengineering. DNA ligase The fragments are able to join together under the influence ofDNA ligase.

10. DNA Amplification PCR – polymerase chain reaction

11. DNA Amplification • Using the technique called polymerase chain reaction (PCR), researchers are able to create vast quantities of DNA identical to trace samples. This process is also known as DNA amplification. • Many procedures in DNA technology require substantial amounts of DNA to work with, for example; • DNA sequencing • DNA profiling/fingerprinting • Gene cloning • Transformation • Making artificial genes • Samples from some sources,including those shown here,may be difficult to obtain inany quantity. A crime scene (body tissue samples) A single viral particle (from an infection) Fragments of DNA from a long extinctanimal

12. Essential PCR Tools • Taq Polymerase – an enzyme that works well at 72oC • Primers • Primers are synthetic short segments of DNA up to 25 nucleotides long.

13. PCR Equipment Amplification of DNA can be carried out with simple-to-use PCR machines called thermal cyclers (shown below). Thermal cyclers are in common use in the biology departments of universities as well as other kinds of research and analytical laboratories.

14. Steps in the PCR Process The laboratory process called the polymerase chain reaction or PCR involves the following steps 1-3 each cycle: Separate Strands Separate the target DNA strands by heating at 98°C for 5 minutes Incubate Cool to 60°C and incubate for a few minutes. During this time, primers attach to single-stranded DNA. DNA polymerase synthesizes complementary strands. Add Reaction Mix Add primers, nucleotides (A, T, G and C) and DNA polymerase enzyme. Repeat for about 25 cycles Repeat cycle of heating and cooling until enough copies of the target DNA have been produced.

15. Polymerase Chain Reaction Original DNASample • Although only three cycles of replication are shown here, following cycles replicate DNA at an exponentialrate and can make literally billions of copies in only a few hours. • The process of PCR is detailed in the following slide sequence of steps 1-5. Cycle 1 Cycle 2 Cycle 3

16. The Process of PCR 1 A DNA sample called the target DNA is obtained DNA is denatured (DNA strands are separated) by heating the sample for 5 minutes at 98C Primers (short strands of mRNA) are annealed (bonded) to the DNA Primer annealed

17. The Process of PCR 2 The sample is cooled to 60°C. A thermally stable DNA polymerase enzyme binds to the primers on each side of the exposed DNA strand. This enzyme synthesizes a complementary strand of DNA using free nucleotides. After one cycle, there are now two copies of the original sample. Nucleotides Nucleotides