1
Q
What is Genetic Engineering?
A
- = the manipulation of the DNA sequences of an organism
- Genetic Code is UNIVERSAL, using same 4 bases – A, T, C & G.
- Same codons code for same amino acids in all organisms, allows genetic information to be transfered between species!
2
Q
For GM Insulin Bacteria, how to produce cDNA from mRNA [isolation]?
A
- mRNA is identified and isolated from β-cell, from nucleus
- Using free nucleotides and enzyme Reverse Transcriptase, undergo reverse transcription on single-stranded mRNA of insulin production, as mRNA as template
- single stranded cDNA produced, now undergoes DNA Replication to produce DOUBLE-stranded cDNA!
3
Q
For GM Insulin Bacteria, how to insert gene into VECTOR [insertion]?
A
- Vector used = bacterial PLASMIDS!
- using Restriction Enzymes/Endonucleases: special enzymes used to form staggered Sticky Ends cutout on DNA Base sequence, to allow gene insertion
- Sticky Ends = complementary cutout of DNA bases, to allow re-joining of base sequence
- In free nucleotide presence,
- SAME restriction enzyme used for Sticky Ends in plasmid cutout AND gene isolation cutout complementary to sticky ends of plasmid, DNA Ligase forms bonds of sugar and phosphate groups, forming sugar-phosphate backbone!
4
Q
For GM Insulin Bacteria, how to allow GM plasmid to enter bacterial cell [transformation]?
A
- Electroporation: applying HIGH pd/voltage to bacterial cell, distrupting phospholipid bilyer (increasing permeability to take up GM plasmid)
- Heat Shock: applying HIGH temp (and COLD temp) in Ca2+ ion presence, increasing also permeability of membrane!
5
Q
Assesing + Identifying if bacteria has taken up GM Insulin Plasmid successfully??
A
- non-recombinant bacterial plasmids: contains resistance gene for ampiliclin AND tetracycline antibiotic
- recombinant bacterial plasmids: contains resistance gene for ONLY ampiliclin antibiotic, tetracycline antibiotic gene is DISTRUPTED due presence of containing insulin-producing GENE!
- BOTH bacterial types will contain ampilcillin resistance initially, if PLASMID has been taken up….
- FISRT: Grow bacteria in Ampicillin Medium: identifies bacteria taking up ANY plasmid at all (including wanted bacterial insulin recombinants)
- NEXT: undergo Replica Plating of surviving bacterial colonies, onto Tetracycline Medium: dying colonies show successful, insuling-containing plasmid bacteria recombinants!
- Replic Plating: prevents loss of wanted bacteral colonies with insulin-producing gene when dying in Tetracycline Medium!
- SAME LOGIC: works for Flourescent Protiens, over antibiotic resistance (faster, more efficient process..)
6
Q
What is PCR?
A
- Polymerase Chain Reaction!
- used to ampifly short DNA base length, into exponential growth of MANY replicated copies!
- uses samilar principles of DNA Replication, in organisms…
7
Q
What tools are required in PCR, and WHY?
A
- Taq Polymerase: DNA polymerase specifically able to function under high tempratures, and changing of temps (enzyme from extremophile)
- 2 primers: single-stranded DNA strands complamentary to base pairs,from 3’ to 5’ direction)
- Starting region for Taq polymerase to occur in 5’ to 3’ direction, AND prevents DNA to re-join mid PCR……
8
Q
How to undergo PCR technique?
A
- DNA molecule heated to 95’C, breaking hydrogen bonds between comp base pairing, denaturing the DNA double helix molecule
- Cooled to 60’C, where DNA 2 primers anneal to 3’ end ot DNA splitted single strands, for Taq polymerase to attach + prevents DNA rejoining
- Taq Polymerase catalyses addition of free nucleotides and phosphodiester bonds formed, starting at primer!
- New copy of DNA produced, now repeated CHAIN REACTION to allow exponential growth in no. of DNA molecules produced…….
9
Q
Applications of PCR in real-world?
A
- Mutation detection: assessing if certian recessive alleles are present to assess likelyhood of genetic disease
- Forensics: smalll DNA sample amplified to identify individuals present at crime scene…
- Reseach purposes: sequencing DNA of exctinct species, for analysis of base sequence..,
10
Q
How to undergo Electrophoresis?
A
- Extraction: extract DNA from cell sample, and amplify DNA via PCR technique, to obtain more DNA molecules
- Digestion: add many restriction endonuclease enzymes to DNA sample, allowing DNA to be cut down (diff ppl = diff num of DNA cut fragments)
- Separation: DNA cut fragments in gel medium, DNA actracts towards pd applied +ve anode (as DNA is -ve charge due to PO43- groups)
- Where heavy DNA fragments = less distance with MORE resistance moved [and vice versa]
- Use alkaline solution to DENATURE DNA (breaking H bonds between bases) to form single strands…
- DNA Gene Probe = single stranded DNA with radioactive/flourescent marker!
- Southern Blotting: DNA Gene probe is ANNEALED to these single stranded DNA, where radioactive probe and X-ray / UV Light / GFP flourscent protien to view sample
11
Q
How to specifically undergo Radioactive/UV Southern Blotting in Electrophoresis?
A
- Radio: nylon filter paper/nitrocellulose paper placed over gel surface, after alkaline solution added to denature the DNA for single stranded gene-probing
1. DNA fragments on gel surface will blot onto paper, where photographic film is placed OVER this paper
2. Radioactive traces can be viewed on film, due to radioactive gene probes used! - UV: simple gene-probing process, as flourescence can be easily viewed with just UV light!
12
Q
Electrophoresis for Amino Acids?
A
- Same process as for DNA Elctrophresis!
- May be used to assess different types of protien coding for same function (e.g. haemoglobin for sickle cell vs normal cell)…
- Electrophoresis ONLY SEPARATES fragments based on FRAGMENT SIZE, and NOT CHARGE DIFFERENCE (all fragments must be -ve charged, moving in same direction!)
13
Q
Applications of DNA Profiling/Electrophoresis?
A
- Forensic Investigation: identifying crinimals via DNA present in crime scene
- Maternity/Paternity Identification: determining of mother/father hood of child
- Disease Analysis: ssesing protien AA sequencing and effect with possible disease (e.g.Huningtons…)
14
Q
Define DNA Sequencing
A
- determining the BASE SEQUENCE of an organism’s genome
- NOT same as DNA profiling, where here DNA base sequence is compared to known source, to identify certian genome
15
Q
How to undergo classic Sanger DNA Sequencing?
A
- Acquire DNA molecule and DENATURE, FRAGMENT and AMPLIFY with high temp (just like PCR…)
- DNA primers will ANNEAL to DNA single-strands (again, like PCR…)
- Taq DNA Polymerase allows phosphodiester bonds to form between adjacent nucleotides, where free nucleotides bind via Complamentary Base Pairing
- Dideoxyribonucleotides [where both -OH groups removed from pentose sugar] used as part of “free nucleotide mix”
- WHEN these, are incorperated into DNA via Taq, phosphodiester bond forming TERMINATES = [at different fragment lengths]
- Base type (A,T,C,G) nucleotide terminated ON recognised in DNA fragment, after gel electrophoresis occurs!
16
Q
Define Bioinformatics, and its specified USES in biology!
A
- storing/access for using large amounts data, applied for biological research
1. Access to large databases of UNIVERSAL DNA base/AAs/Genome sequences, with Protien Structure and Metabolic Pathway reactions
2. Rapid sharing/retrieving of biological info, via algorithimic uses
3. Identifies disease outbreaks, vulnerable target populations for vaccines
4. Statistical Tests able to be performed…
17
Q
Define Computational Biology, and its specified USES in biology!
A
- analysis of large data amounts, applied for biological research
1. Access to large databases with rapid data processing, with Protien Structure Modelling and AA sequencing
2. Phylogeny/Evolutionary Relationships: comparing genomes ACROSS species/reclassifying species, using aid of large databases…
3. Rapid sharing/retrieving of biological info, via algorithimic uses
4. Statistical Tests able to be performed…
