Techniques in biotechnology

1. Techniques in biotechnology Week 3 (26 September 2013) Department of Chemical Engineering Technology, UniMAP. khadijahhanim@unimap.edu.my

2. Course outcome • CO2: • Able to demonstrate important recent advances in methods and applications of biotechnology with regards to microorganisms and plants. • Topic covers: • Techniques in Biotechnology - Demonstrate the basic principles of Recombinant DNA Technology and illustrate other methods used in biotechnological field, to include tissue culture, electrophoresis, Polymerase Chain Reaction (PCR) and biosensor

3. Biotechnology today • Focuses on DNA Deoxyribonucleic Acid- a double-stranded helical molecule that stores genetic information for the production of all the organism’s proteins

4. Review on cell structure • Cells are structural and functional units of all life-forms. • Cells are diverse • BUT they share a common component- genetic information in the form of DNA • Genes contained within DNA control numerous activities in cells by directing the synthesis of proteins. • Genes control behavior: determine skin, hair, eye color and affect our susceptibility to genetic diseases.

6. Evidence that DNA is the inherited genetic material • It was first provided by microbiologist Frederick Griffith 1928.

7. This exp has provided that the genetic material from heat-killed S cells has transformed R cells into S cells. • This exp has demonstrated transformation; uptake of DNA by bacterial cells. • Transformation- powerful techniques in molecular biology and used to introduce genes into bacteria for DNA cloning. • Griffith hypothesized that some genetic factor that responsible for the transformation but did not identify DNA as genetic factor.

8. In 1944, Avery, MacLoed and McCarty provide the evidence for DNA as genetic material.

9. DNA Structure • Erwin Chargaff did a chemical analysis of DNA from different species. • He revealed that the percentage of bases adenine = thymine and cytosine = guanine. • Nucleotides – building block of DNA • Nucleotide composed of pentose sugar, phosphate and nitrogenous base

10. Watsons and Crick discovered the DNA structure in 1953. • Nucleotides are joined together to form a long strands and each DNA molecule consists of 2 strands that wrap around each other to form right-handed double helix. • Held together by phosphodiester bonds that connect sugar molecule of one nucleotide with adjacent phosphate group of one nucleotide. • The two strands are joined together by hydrogen bonds between complementary base pair.

12. Genes, chromosomes and Genomes

14. Recombinant DNA Technology • Recombination DNA technology: cutting and pasting DNA from different source. • Recombination DNA technology is a technique used to make gene cloning possible. • Genetic engineering using the techniques in recombinant DNA technology and gene cloning to modify an organism’s genome.

15. Restriction enzymes and Plasmid DNA Vectors • The discovery of restriction enzymes and plasmid DNA made recombinant DNA possible.

16. Restriction enzymes

17. Plasmids

18. Transformation of Bacterial Cells • Transformation is process of inserting foreign DNA into a host for replication purposes. • Transformation: treated with CaCl2 solutions, add plasmids to cells and chilled on ice, then heat-shock the mixture. • Once inside cell, plasmid will replicate and express their genes. • Modern transformation technique: electrophoration involves brief pulse of high voltage electricity to create tiny holes in the bacterial cell wall that allow DNA to enter.

20. Screening of recombinant bacteria

22. Types of Vectors

23. How do you identify and clone gene of interest? • Many cloning strategies begin by preparing a DNA library. • DNA library- a collection of cloned DNA fragments from a particular organism contained within bacteria or viruses as a host. • Libraries can be saved for long periods and screened to pick the gene of interest.

24. Genomic Libraries • Chromosomal/genomic DNA from the tissue of interest is isolated and digested with a restriction enzyme.

25. Disadvantages of genomic libraries • For eukaryotic genes- noncoding pieces of DNA called introns are cloned in addition to coding sequences (exons). • Since eukaryotic DNA consists of many introns, many of the clones will contain non-coding pieces of DNA • Eukaryotic has a large genome therefore to find a gene of interest from genomic library are laborious. • Therefore, cDNA library are created.

26. cDNA libraries

27. Library screening

28. Polymerase Chain Reaction • Although libraries are very effective and commonly used for cloning and identifying a gene of interest, the polymerase chain reaction (PCR) is more rapid • PCR was develop in mid 1980s by Kary Mullis • A technique used to make more copies of DNA in-vitro • Requires all of the building blocks of DNA • Deoxyribonucleotide, dNTPs (dATP, dTTP, dATP and dGTP) • DNA polymerase • A pair of primers • DNA template

29. What is a primer? oligonucleotide • A primer is a short oligonucleotide which is the reverse complement of a region of a DNA template. • It would anneal to a DNA strand to facilitate the amplification of the targeted DNA sequence.

30. Thermal Cycler machine • An instrument used to do PCR. • Sophisticated heating block that is capable to rapidly changing temperature over a short time.

31. Stages of PCR

33. Polymerase Chain Reaction:One PCR Cycle 90 °C 50 °C 72 °C Taq Polymerase Primer OriginalDouble-helixDNA SeparateDNAStrands Primers &Taqpolymerasebind DNAsynthesized

34. Functions of PCR • Making DNA probes • DNA cloning • Studying gene expression • Amplifying minute amounts of DNA into detectable amount • DNA sequencing • Diagnose genetic disease • Detect pathogens

35. Applications of Recombinant DNA Technology • Study gene structure, sequence and function • Study gene expression • Scale up production of therapeutic proteins • Use in human gene therapy • making antibodies/vaccines for medical purposes • Creating genetically engineered microbes/plants • Use in forensics applications

36. Gel Electrophoresis • Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Agarose gel electrophoresis is routinely used for the preparation and analysis of DNA. • Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. • Using agarose gel electrophoresis to determine the presence and size of PCR products/cloned fragments.

38. DNA sequencing • After a gene is cloned, it is important to determine the nucleotide sequence of a gene. • Exact order of As, Ts, Gs and Cs. • Knowing DNA sequence can be helpful in: • To deduce amino acid sequence of a protein encoded by a cloned gene • To determine the exact structure of gene • To identify regulatory elements such as promoter sequences • To identify genetic mutations

39. DNA sequence method was first developed by Frederick Sanger in 1977. • The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleotidetriphosphates (dNTPs), and modified nucleotides (dideoxyNTPs) that terminate DNA strand elongation.

41. Genomics and Bioinformatics: hot new areas of biotechnology • With the rapid and advances in cloning and sequencing technologies- led to the use of strategies for cloning, sequencing and analyzing the entire genome – field called genomics. • Shotgun cloning- clone and sequence the entire genome. • In shotgun cloning, entire genome, introns and exons is cloned and sequenced then they are sorted using bioinformatics.

43. Bioinformatics • After cloning and sequencing a newly identified gene or DNA sequence, findings are reported in scientific publications and submit the sequence to databases so that others can access the sequence. • Bioinformatics involves the use of computer hardware and software, organize, share and analyzed data related to gene structure, gene sequence and expression and protein structure and function. • The databases generated are essential tools for archiving and data sharing • When a scientist who have cloned a gene enter their sequence data into database, these databases search the new sequence against all other sequences in the database and create alignment of similar nucleotide sequences if it finds a match.