BCM302 Food and Beverage Biotechnology Topic 3: Basic Principles of Recombinant DNA technology

BCM302 Food and Beverage Biotechnology

Topic 3:Basic Principles of Recombinant

DNA technology

Learning objectives1. Know the function of restriction endonucleases, how they work to

cut DNA, and why they are important in biotechnology. Compare blunt ends with sticky ends.

2. Know the mechanism by which electrophoresis separates pieces of DNA.

3. List and know the steps of DNA cloning. 4. Know how vectors are used to transform bacteria, and know the

methods of selecting for successfully transformed bacteria. Compare the types of vectors in terms of the sizes of DNA that can be carried by the vector into the bacteria.

5. List the types of vectors that can be used to transform yeast mammalian cells and plants, and why they are effective in those organisms.

6. List the methods of transformation of cells.

Learning objectives

7. Compare genomic libraries, cDNA libraries, and expression libraries in terms of how they are constructed, what the libraries are looking for, and how they are screened.

8. List the various types of reporter genes used in research.

9. Compare Northern and Southern blot hybridization in terms of how they are constructed and what each type of hybridization is looking for.

10.Know the function of PCR, the steps of PCR, and what PCR allows researchers to accomplish.

11.Compare the two methods of DNA sequencing: the chemical method and the Sanger method, and know which method is more widely used. How does automation impact DNA sequencing?

12.List and define the various methods of analyzing proteins. Are any of these methods similar to DNA methods?

13.Know the types of microarrays, and how DNA and protein microarrays work.

14.List the applications of recombinant DNA technology.

Recombinant DNA Technology: Promise and Controversy

• What is DNA cloning?• Applications:

– New medical diagnostics and treatments– Better vaccines– Stress-resistant crops– More nutritious food– Healthier livestock– Cleaner environment

Recombinant DNA Technology: Promise and Controversy

• Criticisms:– Where to draw the line (human cloning,

organs for donation, selecting characteristics in engineered children)

– Effect on health, hidden dangers, environmental pollution (eg escaping genes and organisms)

– Tampering with the “natural world”

• Solutions?:– Open communication

Cutting and Joining DNA

• Recombinant DNA: When two pieces of DNA are joined together to form a new DNA molecule

• Cloning: Insertion of DNA molecules in bacteria so that many identical copies of the DNA are made

• Gene expression: DNA can be transcribed and protein can be translated within cell

Restriction enzymes

• Also called restriction endonucleases

• Cut DNA at “restriction sites or “restriction sequences”

• Cut across the sugar-phosphate backbone of DNA

Restriction enzymes

• Palandromic, Four-six bases in length

Restriction enzymes

• Derived from bacteria• Natural role: defence against

bacteriophage• Bacterial DNA protected by methyl

groups on adenine or cytosine

Restriction enzymes

• Blunt ends: enzyme cuts directly across the two strands

• Sticky ends: enzyme cuts strands in different places, leaving a short single stranded piece of DNA hanging over the end of the molecule

Restriction enzymes

• Blunt vs Sticky

Separating restriction fragments

• Digestion of DNA with Restriction enzymes may result in fragments of different sizes which need to be separated

• DNA passed through a “molecular sieve” to separate molecules of different sizes

Agarose gel

• Agarose: polysaccharide derived from seaweed

• Powder mixed with buffer, heated and forms a solid gel when cooled

• “Wells” formed in gel during cooling for sample loading

Electrophoeresis

• Electric charge applied to allow DNA to migrate (towards +ve electrode)

• DNA slightly negative due to sugar-phosphate backbone• Large molecules migrate slower• Small molecules migrate faster• Size of pores determined by Agarose concentration

– Lower % = larger pores = better separation for larger molecules– Higher % = smaller pores = better separation for smaller

molecules

Visualizing DNA

• Ethidium bromide: binds to DNA and fluoresces when exposed to UV light

• Size of DNA fragments determined by comparing with fragments of know sizes

• DNA fragments migrate at a rate that is inversely proportional to the logarithm of the size of the fragment (in base pairs)

Agarose gel Electrophoresis

Acrylamide gel electrophoreis

• Gel formed using polyacrylamide

• Vertical• Used for smaller

molecules (eg DNA sequencing)

DNA cloning

• Isolation of DNA• Ligation of the DNA to a vector• Transformation of a host cell with

the recombinant DNA• Selection of host cells containing

the DNA

Isolation of DNA

• Cleaved from a larger piece of DNA• Generated using the Polymerase

Chain Reaction (see later)

Ligation

• DNA ligated to a cloning vector

• Enzyme: Ligase• Requires

compatible ends

Cloning Vectors

• Transport molecule so DNA can be replicated in cell:

• Features:– Origin of replication (Ori)– Small– Multiple cloning site (MCS) with unique RE

sites– Selectable marker (to determine which

bacteria contain the vectors with the inserted DNA)

Bacterial vectors (Plasmids)

• Extrachromosomal pieces of DNA not necessarily needed by bacteria

• Natural roles eg antibiotic resistance pigment production

• Engineered to accept DNA fragments up to 10 kilobases in length

• High copy number and low copy number

Plasmid: pBR322

• Contains unique REs

• Antibiotic resistance (to determine which bacteria contain vectors)

Plasmid: pUC18/19

Alpha complementation

• lacZ gene codes for beta-galactosidase

• Beta-galactosidase breaks down X-gal to produce a blue product

• If molecule cloned into MCS (in lacZ gene) X-gal will not be broken down and colony will be white

Colony selection

Other vectors

• Bacteriophage – virus that infects bacteria

• Cosmids – Has aspects of both plasmid and bacteriophage

• Yeast Artificial Chromosome (YAC)– Contains centromeere, telomere, autonomously

replicating sequence (ARS), selectable marker gene

• Bacterial Artificial Chromosomes (BAC)– Created using a small plasmid with a F (fertility)

factor that allows the vector to accommodate larger pieces of DNA (up to 25% of the size of the bacterial Chromosomes)

Plant Cloning Vector

• Tumor inducing (Ti) plasmid

• Replicates in both E.coli. and Agrobacterium

• Facilitates plant transformation

Cell transformation

• Transformation: insertion of DNA into a cell

• Methods:– Calcium Chloride/heatshock (Bacteria)– Electroporation (Bacteria, mammalian,

protoplasts)– Microinjection (Mammalian)– Biolistics (Plant)

Plant Transformation

• DNA coated onto small particles (eg gold or tungsten)

• Accelerated using a particle gun

DNA Libraries

• Used to help map genomes• Can be screened to find specific

genes• Different types:

– Genomic library– cDNA library

Genomic Library

• Digest genomic DNA and plasmid

• Ligate DNA to vector

• Transform bacteria with recombinant vectors

• Screen

cDNA Library

• A DNA library constructed from cDNA

• cDNA (complementary DNA): a DNA copy of messenger RNA (mRNA)

cDNA synthesis

• Pre-mRNA is transcribed by cell

• Introns are removed

• A single stranded cDNA copy is made using reverse transcriptase

cDNA synthesis (con’t)

• mRNA is degraded

• Second DNA strand synthesised using DNA polymerase

cDNA Library

• cDNA can be derived from RNA isolated from specific organism, tissue or treatment

• cDNA ligated to vector as per genomic DNA

Screening libraries

• Bacterial colonies can be screened for the presences of specific:– DNA (southern hybridisation)– RNA (northern hybridisation)– Proteins (western hybridisations)

Colony Hybridisaton

• DNA transferred to membrane• Membrane probed with complementary DNA

Probe hybridisation

• DNA probe with complementary sequence will bind to DNA from colony

Reporter genes

• Connected to gene of interest to study expression pattern

• Eg: Luciferase, GFP, GUS

Southern Hybridisation

• Invented by Edward Southern in the mid-1970s

• Allows the detection of a DNA fragment in a large population of molecules


• Steps:– DNA fragments can be separated on a gel and

denatured– Fragments transferred to a nylon or nitrocellulose

filter– Radioactive DNA probes hybridised to membrane– Position of radioactivity (detected by

autoradiography) indicates position of DNA of interest




Northern Hybridisation

• Similar to Southern hybridisation except RNA used instead of DNA

• Used to assess levels of gene expression

Western hybridisation

• Similar to Southern and Northern Hybridisation except it involves detection of proteins with antibodies

Polymerase chain reaction (PCR)

• Amplification of specific pieces of DNA

• Three steps (cycles) repeated 25-40 times:– Denaturation (Double stranded

melted)– Annealing (primers bind to specific

seq.)– DNA synthesis (DNA replicated)

Polymerase chain reaction

Polymerase chain reaction

• Primers: – Short pieces of single stranded DNA (oligonucleotides)

complementary to sequence to be amplified

• DNA polymerase: – Enzyme that can

synthesise DNA suing a DNA template

– Requires double stranded molecule to initiate synthesis

Steps in PCR

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Applications of PCR

• Isolate specific DNA for further analysis

• Generate DNA fingerprint to determine genetic relationships

• DNA sequencing

DNA sequencing

• Two types:– Chemical method: Selective

degradation– Dideoxy method: generates

fragments of different sized during replication by interrupting synthesis

DNA sequencing



DNA sequencing



Protein Gel Electrophoresis

• Two types of gels:– One dimension– Two dimension

1-D Gel electrophoresis

• Vertical polyacrylamide gels• Contains sodium dodecyl

sulphate (SDS) to interrupt hydrogen bonding

• Proteins run based on size• Visualised by staining

(coomassie blue) or radiolabeling



2-D gel electrophoresis• First dimension: proteins

separated based on charge• Second dimension: proteins

separated based on size• Visualised by staining (silver

stain) or radiolabelling• Applications:

– Analysing whole cell protein extracts– Assessing different forms (eg

glycosylated) of a protein



Protein engineering

• Protein sequence changed by altering the DNA sequence

Applications of Protein engineering

• Applications– Resistance to degradation, pH

change, temp., oxidation– Enhance activity– Change substrate specificity– Increase nutritional value

Protein sequencing

• Edman degradation method• Mass spectrometry

DNA microarray technology

• Whole genome analysis• Can compare all genes expressed

in diff. tissues/diff. Conditions



Microarray procedure

• Step 1: Prepare array




• Step 2: Preparing and hybridizing labelled target cDNA




• Step 3: Analysis

RNA interference technology

Additional resources

• www.protocol-online.org

• Protocol Online—a searchable list of laboratory protocols in molecular biology, cell biology, biochemistry, and proteomics.

• www.biology.arizona.edu/molecular_bio/problem_sets/Recombinant_DNA_Technology/recombinant_dna.html

• Recombinant DNA Technology Problem Set—questions dealing with PCR, recombinant DNA technology, and Southern blot analysis, along with applications of these techniques.

• web.mit.edu/esgbio/www/rdna/rdnadir.html

• Recombinant DNA—descriptions of DNA cloning techniques, hybridization techniques, PCR, and DNA fingerprinting technology. This site also contains problems that test your knowledge of these techniques.

• www.bio.davidson.edu/courses/genomics/chip/chip.html

• DNA Microarray Methodology Animation—an animated description of DNA microarray experiments.

• webphysics.davidson.edu/applets/biogel/biogel.html

• Gel Electrophoresis Simulator—a Java applet that allows you to enter the sizes of DNA pieces and see where they may travel on an electrophoresis gel.

• arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/virgel.html

• Virtual Lab: Agarose Electrophoresis—s Java applet simulation of DNA gel electrophoresis that allows you to start and end electrophoresis, as well as see a gel as it might appear under ultraviolet light.

• arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/

• Gel Electrophoresis of DNA and RNA—in-depth descriptions of the concepts behind gel electrophoresis of nucleic acids.

• us.expasy.org/ch2d/protocols/

• Technical Information on 2-D PAGE—a web site dealing with the theories behind 2-dimensional gel electrophoresis of proteins (2-D PAGE).

• http://www.cc.ndsu.nodak.edu/instruct/mcclean/plsc431/cloning/clone3.htm

• Cloning and Molecular Analysis of Genes—a description of the types of vectors used in DNA cloning.

Additional resources

• www.dnalc.org/shockwave/southan.html

• DNALC: Southern Blotting—an animated tutorial of the technique of Southern blot analysis.

• www.dnalc.org/shockwave/pcranwhole.html

• DNALC: PCR Animation—an animated tutorial of the theory and steps of the Polymerase Chain Reaction (PCR), along with an example of PCR in action.

• www.ndsu.nodak.edu/instruct/mcclean/plsc431/cloning/clone5.htm

• Clone Library Screening—a short description on how DNA and genomic libraries are screened.

• www.ascb.org/teachers/green.html

• Green Fluorescent Protein (GFP) Links for Teachers—hosted by the American Society for Cell Biology, this Web site provides information regarding one of the more popular reporter proteins used in biotechnology research.

• www.biotechniques.com

• BioTechniques—a monthly magazine devoted to covering new developments regarding techniques in biotechnology.

• www.promega.com/biomath/default.htm

• BioMath Calculators—a Web-based series of calculators for common calculations in molecular biology, such as the melting temperature of short pieces of DNA called oligos, conversion of absorbance to concentration of DNA in a sample, and dilution calculations. Hosted by Promega Corporation.

• homepages.gac.edu/~cellab/index-1.html

• Cell Biology Laboratory Manual—a collection of laboratory protocols in cell biology, along with a list of links to other Web sites related to cell and molecular biology.

• www.carolina.com/biotech/Default.asp

• Carolina Biological: Biotechnology and Genetics—hosted by Carolina Biological Supply Company, this Web site provides information and suggestions for presenting various topics in biotechnology to students. Links to other Web sites, as well as video clips are also available.

Documents

BCM302 Food and Beverage Biotechnology Topic 3: Basic Principles of Recombinant DNA technology