Biology Application Syllabus: Isolating, Cloning and Sequencing DNA1

1. Describe the natural function of restriction enzymes (RE).

They are mainly from bacteria. Used to cleave invading foreign DNA molecules (bacteriophage) Note that bacteria cells own DNA is not cleaved as the restriction sites recognized by the restriction enzymes are methylated. (Methylation by methylase protects host DNA from cleavage) (They add methyl groups to A or C residues)

2. Explain the formation of recombinant DNA molecule.

Cleaving at restriction sites RE recognize specific base sequences in double-stranded DNA and cleave both strands of the DNA at specific places. They recognize and target specific sequences of 4 6 base pairs. They will cut DNA at every point at which its target sequences occurs, by hydrolyzing phosphodiester bond in each strand of DNA, producing two d-s DNA fragments. (which gives rise to many DNA fragments) Restriction sites have twofold rotational symmetry such that the same 5 3 sequence is found on both strands but in opposite direction (sequence is palindromic) Note: difference between sticky ends and blunt ends. D-s DNA fragments with protruding ends (cohesive/sticky ends) have these short extensions that anneal (through H-bonding) with complementary s-s stretches on other DNA molecules cleaved with the SAME restriction enzyme. Blunt/flush ends do not normally bind specifically to any other blunt ends. Thus, less specificity in binding. Thus, need for terminal transferase, to add dCTP at 3 ends of the d-s DNA strands, and dGTP at 5 ends on the linearised plasmids. The strands can then be sealed using DNA ligase.

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3. Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid and
describe the properties of plasmids that allow them to be used as DNA cloning vectors. Procedures for cloning gene in bacterial plasmid Step 1: Isolate DNA from two sources Lyses cell, purification and concentration of DNA. Extract plasmids (vectors) from bacteria cells. (Or obtain commercially) Step 2: Form recombinant DNA molecules Restrict both with same RE to leave complementary sticky ends. (DNA sample produces many restriction samples) Mix DNA and allow sticky ends to anneal. Ligate. Step 3: Transform competent bacterial host cells with the ligation mixture (containing the desired recombinant plasmid) Transformation is the process where a host cell assimilates external DNA, resulting in genotypic and phenotypic change. (When these cells grow and divide, the recombinant plasmids inside them replicate to produce the copies of genes of interest) To facilitate transformation, host cells are rendered transiently permeable become competent cells. Transformation is not efficient. (Cells are either not competent enough or killed during process) Method Ca2+ treatment and a brief 42C heat shock CaCl2 cause DNA to precipitate on the outside of the cells. Actual uptake stimulated by brief heat shock (Other methods: Electroporation, Liposome, Microinjection, Viral Injection) Step 4: Screening for correct transformant.

(Screen for host cells transformed with recombinant DNA molecule containing insert of interest) Vector DNA usually have antibiotic resistant genes as selectable marker Step 4a: Screen for vector DNA. E.g. -lactamase gene confers resistance to ampicillin. Cells which have taken up the plasmid will survive on agar plate containing ampicillin. Mixture appropriately streaked such that colonies that grow originate from one bacterial cell. Cells that can grow on the plate must have taken up the vector DNA Step 4b: Screen for recombinant plasmid When the gene of interest was inserted, it should have disrupted a second selectable marker. Replica-plating Gently press a velvet pad onto the original master plate (with ampicillin) Press the pad onto a second plate containing the second selective agent (i.e. tetracycline) Only the recombinants loses tetracycline resistance. Cells which grew on master ampicillin plate but not on tetracycline plate are successful recombinants. Step 5: Large scale production Inoculate bioreactor (which has nutrient broth and impeller blades to circulate the solution) with transformant Extraction and purification

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Properties of plasmids as DNA cloning vectors Why are linear DNA fragments as vectors are not desirable? May not carry any sequence that specify replication by host cell DNA polymerase Susceptible to degradation by DNAse Plasmids have origin of replication (ori) (50-100 bp) enable host cell polymerase to initiate replication autonomous replication. Copies of recombinant DNA molecules can thus be produced and passed on. Plasmids have high copy number per host cell. Large quantities of the recombinant DNA molecule can be obtained from each cell. Plasmids have selectable markers Genes that confer readily selectable phenotypic traits (e.g. resistance to antibiotic) Gene of interest is inserted in between one of the selectable markers (insertional inactivation) Most contain at least two selectable markers One intact to select transformant, and the other to detect inactivation Note: Insert DNA that are too large (>10kb) are not replicated efficiently as under selective pressure, the large plasmids would tend to delete non-essential genes (i.e. foreign inserts)

4. Explain how eukaryotic genes are cloned using E. coli cells to produce eukaryotic
proteins to avoid the problems associated with introns. Bacteria lack: Ability to perform post-transcriptional modifications Ability to perform post-translational modifications Ability to recognize eukaryotes promoter Derive DNA sequences from mRNA instead of genomic DNA, with all introns removed mRNA DNA using reverse transcriptase (DNA complementary to RNA in 5 3 direction) Ligate into cloning vectors

5. Distinguish between a genomic DNA and cDNA library.

Genomic Library Contains entire DNA content Requires chromosomal DNA isolation Can obtained from any tissue Cleaved with RE before ligating into cloning vector Disadvantages Eukaryotic genomic DNA contains introns disallow cloning by prokaryotes(inability to modify pre-mRNA) Large eukaryotic genome too many fragments to screen cDNA library Contains entire protein-encoding DNA content Requires total mRNA isolation Total mRNA should be isolated from tissue where particular protein is likely to be produced in large quantities Reverse-transcribed into cDNA before ligating into cloning vector Advantages No introns eukaryotic genes correctly expressed in prokaryotes Abundance of mRNA in particular tissue type facilitates screening of recombinant clones by increasing chance of obtaining correct clone because these tissues usually contain more of these specific sequences

DNA sequence of gene unknown gene of

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interest may be cut internally

6. Outline 2 important proteins and other products that can be produced by genetic engineering technique (e.g. human growth hormone, anti-thrombin, etc). Human antithrombin III Structure: Glycoprotein with 41 aa, secreted by liver Function: Anticoagulant Inhibits serine proteases that participate in blood clotting cascade Problems: Deficiency leads to thrombosis, embolism Human growth hormone (somatotrophin) Structure: Peptide hormone with 191 aa, secreted by somatotrope cells of anterior pituitary gland Function: Stimulate growth and cell reproduction Problems: Excess may lead to Agromegaly (uncontrolled bone growth), deficiency may lead to dwarfism Human Insulin Structure: Peptide hormone, 2 chains (21 and 30 aa), secreted by -cells of Islets of Langerhans in pancreas Function: Controls level of glucose in blood (You should know more than just this from core syllabus) Problems: Deficiency may lead to diabetes mellitus

7. Describe the polymerase chain reaction (PCR) and explain the advantages and
limitations of this procedure.

PCR used for amplification of a specified segment of DNA Components for PCR: Source DNA containing the segment to be amplified Oligonucleotide primers Synthetic s-s DNA (20-30bp) Initiate DNA synthesis Complementary to seq. flanking target DNA segment (2, one to each strand of DNA double helix) To have them in large excess Taq polymerase Thermo stable Stable at 95C, optimal at 72C Deoxyribonucleoside triphosphates (dNTPs) Substrates for DNA replication (dA/T/C/GTP) Buffer (containing Mg2+) Procedures of PCR Brief heat treatment (up to 95C) to separate DNA double helix Cooling to 64C in presence of excess DNA primers to allow attachment Taq polymerase performs synthesis of complementary DNA strand at 72C (optimal temperature) Each denaturation-hybridisation-synthesis cycle results in doubling the number of DNA sequences replicated. X cycles will yield 2X strands of target DNA. Advantages of PCR Limitations of PCR

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Each round of PCR doubles number of target DNA. Amount of desired sequence increases exponentially Fully automated within thermocycler convenient way to amplify DNA fast and efficiently Highly sensitive target sequence can be amplified even when only a minute amount of DNA source is available Contributes in clinical(detecting genetic disease at embryonic stage), forensic, archaeology and paleontology(amplified to have large amounts to analyze)

Taq polymerase lacks 3 to 5 proofreading ability Success of PCR requires knowledge of the sequences flanking the target region. Incorrectly designed primers will result in failure DNA fragment are limited to ~3kb. Further increase decreases efficiency as the polymerase tends to fall off the DNA before replication is complete

8. Explain how gel electrophoresis is used to analyze nucleic acids and proteins and to
distinguish between two alleles of a gene. Gel electrophoresis is a technique by which charged molecules are separated based on their size or charge, by passing them through a gel within an electric field Agarose Gel Electrophoresis is used for separation of DNA. (Agarose is polysaccharide from seaweed that forms the gel) SDS-PAGE (Sodium dodecyl sulphate polyacrylamide gel electrophoresis) is used for analysis of proteins Procedures for Agarose Gel Electrophoresis A slab of agarose gel is placed in buffer solution which allows conduction of electricity to generate the electric field Gel pre-cast with little indentations (wells) at one end, using a comb. Each well corresponds to one lane. DNA sample mixed with dense loading dye(helps DNA sink to bottom, and monitor progress of electrophoresis) Markers(prepared mixtures of DNA fragments of known size) are run alongside, form basis of comparison. When the current in turned on, -ve charged DNA fragments move towards anode. The meshwork of polymer fibers in agarose gel impedes movement, such that shorter DNA fragments move faster than longer fragments due to less resistance. DNA sample will separate to form discrete bands on the gel Ethidium bromide is used to visualize the bands, under UV light, as the dye bound to DNA fluoresces. Procedures for SDS-PAGE Performed in polyacrylamide gels. Similarly, gel pre-cast with little indentations (wells) at one end, using a comb. Each well corresponds to one lane. Proteins first treated with SDS (anionic detergent), denaturing the proteins (breaking hydrophobic interactions and coating polypeptide with many ve charge, thereby linearising them), thus proteins are separated by mass alone. (Boil with -mercaptoehanol to denature disulphide bonds) Mixture of denatured proteins applied to gel, and apply current. Smaller proteins move faster than larger proteins. Stain with Coomassie Blue to visualize proteins. (Forms silver stain) Procedures for 2-D Gel Electrophoresis The gel is first made to have pH gradient from left to right, with left more acidic

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Protein mixture loaded at pH = 7, voltage applied, causing proteins to move through the gel, until isoelectric point. Subject to SDS-PAGE, apply voltage 90 to the first separation, separating the proteins by mass. (Refer to above) To distinguish between two alleles of a gene Mutation/Differentiation located within a restriction recognition site for restriction enzyme. As a result, restriction enzyme no longer recognizes the site. Different mixtures of fragments will be produced from each allele. For example, point mutation in sickle-cell anaemia causes Mst II, a RE to no longer recognize the restriction site. No restriction takes place, generating 1 instead of 2 fragments.

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9. Outline the process of nucleic acid hybridisation and explain how it can be used to
detect and analyze restriction fragment length polymorphism (RFLP). Procedures of Southern Blotting DNA cut by RE and separated by electrophoresis. Gel slab placed under nitrocellulose membrane and a stack of paper. These are then placed on top of an absorbent sponge in alkaline solution. Capillary action draws alkaline solution upward through the gel, denaturing the d-s DNA fragments. S-s DNA is then drawn onto the nitrocellulose membrane, binding in the exact same position as in the gel. Nitrocellulose membrane removed and incubated with radioactive DNA probe, which are s-s DNA synthesized to be complementary to specific nucleotide sequences Fragments containing these specific nucleotide sequences will hybridize to probe by complementary base-pairing. Membrane is washed to remove unhybridized probes Autoradiography performed by placing an X-ray film over the membrane. Radioactivity of those probes for image corresponding to bands that have bound to the probe.

The whole process of separating DNA fragments by agarose gel electrophoresis, blotting fragments onto membrane filter, hybridizing with labeled complementary probe is called Southern Blotting 10.Explain how RFLP analysis facilitated the process of genomic mapping, diseases detection, DNA fingerprinting, etc.

DNA polymorphisms represent the small nucleotide sequence differences in different individuals. It can differ by either in nucleotide sequence, or having variable numbers of tandemly repeated nucleotide units. Restriction fragment length polymorphisms are generated from these differences in sequence. They are analyzed by Southern Blotting (oh. surprise.) Uses of RFLP analysis In disease detection In coding region single-nucleotide polymorphism(single bp difference) due to e.g. spontaneous point mutation (sickle-cell anaemia) By carrying out Southern blotting, we can detect the presence of disease allele through the characteristic banding pattern that results on the electrophoresis gel. In flanking region In disease phenylketonuria (PKU), RFLP in question results from change in DNA sequence flanking 3 region. By carrying out Southern blotting, different-sized restriction fragments are produced, allowing identification of individuals carrying the disease allele. Limitations in disease detection Not useful when: more than one mutation may have caused that disease Gene has yet been discovered for a disease Disease caused by multiple gene interactions In DNA fingerprinting

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Probes for microsatellites (STRs) and minisatellites (VNTRs) are used. These vary in numbers in individuals. RE that cuts either side of a STR or VNTR locus will produce characteristic banding pattern on agarose gel. The number of repeats is heritable, thus by comparing banding patterns, we can determine how closely related between individuals. (Note: Cannot exactly pinpoint due to genetic variation and if too few bands are compared) In Genomic Mapping Genomic mapping involves building a picture of the arrangement of genes and other genetic markers relative to each other in the genome. Done by calculating recombination frequencies, or using RFLPs 11. Discuss the goals and implications of the human genome project, including the benefits and difficult ethical concerns. The Human Genome Project was a 13-year old effort, coordinated by U.S. Department of Energy and the National Institutes of Health. The draft human genome sequence estimated about 30,000 to 40,000 protein-coding genes. Protein-coding genes accounted for < 2% of DNA in a cell. Goals of HGP

Identifying important elements in DNA sequence responsible for disease-causing

Foundation to understand how genes, proteins and many other molecules work together to develop and regulate complex living organisms Foundation to understand the molecular processes underlying life Foundation to understand our evolutionary origins Benefits from the HGP

In Molecular Medicine

Genes have been pinpoint and associated with various diseases like breast cancer, cystic fibrosis and liver diseases. In the long term, the understanding may lead to significant advances in their management / treatment. In Evolution and Anthropology

Provide insights into evolutionary relationships among various living things

In Forensics

DNA sequencing technologies will allow more RFLP loci to be studied, for precise identification of individuals In biological research

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Studies can now be implemented on a larger scale, as opposed to studying one or few genes at a time. Sharing of research information through the human genome database available on the net. Concerns arising from the project

The ability to diagnose a genetic disorder before any treatment is available may do more harm than good, since it creates risks of social stigmatization and a sense of helplessness.

Justifications for the high cost accrued by the project funds are diverted from addressing environmental and social issues. Risk of genetic discrimination(e.g. by insurance agencies and employers). DNA fingerprinting provides insights to many intimate aspects of an individual, increasing the potential for genetic discrimination.

It could potentially refuel resurgence of eugenics.

Patenting of gene fragments by private biotech firms to monopolize certain gene test markets impede research progress. One could argue that patenting a part of nature violates basic human rights too.