Recombinant DNA technology

1. Recombinant DNA technology BIO3B

2. Revision Draw a label to identify: The template strand The coding strand A nucleotide A hydrogen bond A codon An amino acid Transcription Translation Describe the roles of Ribosomes tRNA RNA polymerase

3. Revision The coding strand Describe the roles of Ribosomes – site of protein synthesis tRNA – carries amino acids to the ribosomes. Anticodons match to the codons on mRNA so the correct amino acid sequence will be built RNA polymerase – enzyme that makes RNA A nucleotide Transcription A hydrogen bond The template strand Translation A codon An amino acid

4. Recombinant DNA technology This is also known as genetic engineering This refers to the processes that allow introduction of new DNA into a cell. This allows the taking of genes from one organism and placing them in the chromosomes of another producing a transgenic organism. The genes can come from individuals of the same species (eg transferring a healthy gene from one person into a person with a genetic disease = gene therapy) or from a different species (eg placing insulin producing gene into a bacteria)

5. Recombinant DNA technology Uses include gene therapy, producing transgenic organisms or GMO -genetically modified organisms (eg bacteria that can produce useful products eg insulin, insertion of pest resistance into plant crops) Products now being produced by this technology include hormones (eg insulin, hGH, FSH), factor VIII or vaccines Some risks and concerns associated with recombinant technology include cost, religious objections (‘tinkering with God’s work’), risk of cross species diseases or diseases resistant to treatment, unknown side effects

6. Genetic engineering Steps involved include • Identifying the genes responsible for a particular characteristic • Identifying the chromosome that carries the gene • Identifying the location of the gene on the chromosome • Identifying the DNA sequence of the gene • Isolating the gene segment or DNA sequence • Copying the DNA sequence • Inserting the gene into the desired organism • Cloning or breeding the organism to produce many individuals with the desired characteristic

7. Recombinant DNA techniques Restriction enzymes are used to cut sections of DNA at specific locations and are necessary because they allow the selection of 1 gene or DNA fragment Ligation refers to joining of the two segments and is necessary because it produces 1 strand of DNA or a plasmid now containing the gene A gene for antibiotic resistance is usually added so that the bacteria with the new DNA can be identified and collected

8. Terms used in recombinant DNA 1 • Vector - Bacterial plasmids, viral phages or other such agent used to transfer genetic material from one cell to another • Phage or bacteriophage - a virus that infects bacteria • Plasmids - Small circular strands of DNA distinct from the main bacterial genome. They are composed of only a few genes and are able to replicate independently within cells

9. Terms used in recombinant DNA 2 • Restriction enzyme - Enzymes that cut strands of DNA at specific sequences of nucleotides. Most are derived from bacteria. • Ligase - An enzyme that is capable of combining two small components of single-strand DNA into one single structure. • Staggered cut - Produced when a restriction enzyme creates fragments of DNA with unpaired nucleotides that overhang at the break in the strands; called sticky ends • Straight cut - Produced when a restriction enzyme makes a clean break across the two strands of DNA so that the ends terminate in a base pair; called blunt ends • Sticky ends - The overhanging ends produced by a staggered cut of a sequence of nucleotide bases; sometimes called cohesive ends • Blunt ends - The ends produced by a straight cut of a sequence of nucleotide bases

10. Transfer and cloning of insulin gene

11. Gel electrophoresis Gel electrophoresis refers to a process that allows identification or observation of DNA patterns Uses include identification of • individuals from samples (eg forensic science) • genome sequences • presence of inherited disease • species from tissue samples eg checking tuna doesn’t contain whale, checking fish or meat is the species it is sold as

12. Sequencing by electrophoresis

13. Uses of electrophoresis

14. Polymerase chain reaction Polymerase chain reaction (PCR) refers to the process used to replicate DNA. DNA is denatured or split by heating. Primers are attached to the DNA strands and start the replication process This process is repeated many times – doubling the DNA each time This increases the amount of DNA present in a sample. It is needed so that there is enough DNA present to test or use

16. DNA microarrays or chips A microarray allows scientists to carry out tests on many genes at the one time. It works by looking at mRNA molecules which bind specifically to DNA fragments that are the same as template from which it was made. This is called hybridisation. If mRNA attaches to that fragment, it indicates that that gene is active in the tissue from which the mRNA is taken. The array is set up with many DNA samples, each representing a gene. Scientists can determine, in a single experiment, the expression levels of hundreds or thousands of genes within a cell by measuring the amount of mRNA bound to each site on the array. With the aid of lasers and a computer, the amount of mRNA bound to the spots on the microarray is precisely measured, generating a profile of gene expression in the cell. This technique allows the identification of active genes in different tissues eg in cancer cells, or individuals with a particular disease. It is being used in design of treatments and production of new vaccines.

17. DNA microarrays 2