U2T4 - Gene Technology

Question 1

Small blood splatter found at crime scene, why only use white blood cells to obtain DNA for amplification?

Answer 1

Red blood cells don’t have nuclei + therefore DNA.

Question 2

Describe the steps of the PCR.

Answer 2

DNA sample heated to 95c, H bonds between base pairs broken so 2 separate strands. Primers added + DNA cooled to 40c-60c, allowing them to anneal to complementary sequences on sample DNA. Ensures strands stay separate + provides double stranded section for DNA polymerase to bind to. Temp raised to 72c + thermostable DNA polymerase adds complementary bases. Each cycle doubles number of copies.

Question 3

What are the 2 methods of cutting up DNA by restriction enzymes?

Answer 3

Hae 3 cuts straight through sugar-phosphate backbone, creating blunt ends. Others (EcoR1) cut sugar-phosphate in staggered way to create sticky ends.

Question 4

What is genetic fingerprinting based on?

Answer 4

All organisms have diff nucleotide sequences (unless identical twins). Most coding DNA is identical to all other individuals. All humans produce identical enzymes for cells to function. Density of bars indicates num of DNA fragments of certain size. Not linked to fragment size.

Question 5

What are the 2 categories of DNA sequences which are different in each individual?

Answer 5

MRSs + SNPs.

Question 6

What are the main stages of DNA profiling/genetic fingerprinting?

Answer 6

Extraction of DNA from sample, digestion of DNA using restriction enzymes, separation of fragments by gel electrophoresis, heating to make DNA single stranded, Southern blotting + incubation with DNA probes + visualisation.

Question 7

Describe the process of extracting the DNA during genetic profiling?

Answer 7

Extracted by breaking down cell membrane + releasing DNA using detergents, creating a DNA precipitate. The sample size is amplified using the PCR.

Question 8

Describe the process of digesting the DNA during genetic profiling?

Answer 8

Restriction endonucleases cut DNA fragments at specific sites of differing lengths. Can leave sticky or blunt ends.

Question 9

Describe the process of heating the DNA during genetic profiling?

Answer 9

DNA is heated to break the hydrogen bonds between bases to make it single stranded.

Question 10

Describe the process of visualisation during genetic profiling?

Answer 10

Excess probes washed away + membrane visuals using x-ray or UV light so DNA can be studied.

Question 11

Why do restriction enzymes cut DNA to different lengths?

Answer 11

The recognition sequences are random.

Question 12

Describe the process of gel electrophoresis during genetic profiling?

Answer 12

Place DNA samples in wells formed in agarose gel, apply electric current + visualise DNA (dye). Longer fragments move shorter distance. Everyone has diff band pattern as recognition sequences vary in spacing due to numbers of MRSs. If radioactively labelled, x-ray used. If it fogs, have combined.

Question 13

What is the basis of genetic engineering?

Answer 13

Can transfer DNA that codes for specific protein into diff cell which can then transcribe + translate cell into functioning protein as DNA is universal.

Question 14

What are the 6 stages of gene transfer?

Answer 14

Isolation of required gene, inserting gene into vector, getting recombinant plasmids into host cells, finding genetically modified bacteria, multiplication of host cells + downstream processing.

Question 15

What are the 2 possible methods of isolating a gene in gene transfer?

Answer 15

Cutting gene out of its DNA chain or using mRNA.

Question 16

Describe how gene is cut out of its DNA chain to isolate a gene?

Answer 16

Restriction endonucleases cut gene out of chromosome. Molecules of DNA probe hybridise to complementary base sequence in cell. Marker indicates where gene is. Once position identified, gene cut out using restriction enzymes at diff base sequences as only they right shape to fit active site.

Question 17

Describe how to isolate a gene using mRNA?

Answer 17

Reverse Transcriptase used. mRNA gotten from cells where gene actively synthesising protein. Carry code for insulin, common in cytoplasm of insulin producing cells in pancreas. Can be used to make artificial insulin genes. (endocrine region containing a + b cells) Human Growth Hormone mRNA found in anterior pituitary cells. mRNA used as template to produce complementary ssDNA from free DNA nucleotides via reverse transcriptase. ssDNA made double stranded using free DNA nucleotides + DNA polymerase, complementary gene made.

Question 18

What is the equation for reverse transcription?

Answer 18

mRNA + DNA nucleotides -Reverse Transcriptase-> cDNA + mRNA

Question 19

Where is reverse transcriptase naturally found?

Answer 19

Retroviruses e.g. HIV, AIDS virus.

Question 20

What are the 2 main advantages of using mRNA to isolate a gene?

Answer 20

Each active cell has many copies of mRNA so easier to find than single copy of DNA gene in each cell + length of mRNA strand corresponds to length of DNA gene. Doesn’t have to be cut out of longer piece, as with DNA.

Question 21

Describe how genes are inserted into a vector.

Answer 21

Once gene isolated, must be inserted into host cell. Easiest way is using vector. Plasmid must be cut out using same restriction enzyme used to cut human gene, produces complementary sticky ends on human gene + plasmid. Genes + plasmids mixed, ligation occurs under right conditions. Controlled by DNA ligase which helps to join DNA of plasmid + gene. Circular DNA molecule produced. Recombinant DNA as from OG plasmid + human gene.

Question 22

Describe the use of viruses as vectors.

Answer 22

Viruses can have donor DNA engineered into own viral DNA. Recombinant viruses can then be fired into host bacterial cells which should then produce protein coded for in viral DNA.

Question 23

Describe how recombinant plasmids are put into host cells.

Answer 23

Plasmids removed from bacterial cells. Incubated with bacterial cells so they take in engineered plasmids. Various treatments make this easier. (calcium ions, temp shock + electrical shock as help to make bacterial cell wall more permeable) Not exact process. Only few bacteria take up plasmid with human gene (recombinant plasmid) Called modified. Other bacteria take up non-recombinant plasmids without human gene.

Question 24

Give an example of a treatment used to make it easier for plasmids to enter host bacteria.

Answer 24

Soak bacteria in ice cold calcium chloride solution, then incubating at 42c for 2 mins.

Question 25

Describe how to find genetically modified bacteria.

Answer 25

Must be screened. May use DNA probes or genetic markers like genes for antibiotic resistance.

Question 26

Describe how genes for genetic resistance are used to find genetically modified bacteria.

Answer 26

Plasmids with gene for antibiotic resistance used as vectors. Plasmid cut + human DNA inserted within bases of antibiotic resistant gene. If inserted successfully, gene can’t function as damaged. Samples taken from OG culture (obtained by replica plating) grown on medium containing antibiotic. Plate out bacteria on ampicillin agar, results in bacteria growing with ampicillin resistance, confirming bacterial colonies that have plasmid. Restriction site used in tetracycline gene so if plasmid has donor gene, won’t confer resistance to host cell. Replica plates made. 1 grown on normal agar, other in tetracycline. Comparison made. Those on normal all contain plasmid, all growing on tetracycline have inserted gene + plasmid, the ones we want.

Question 27

How are host cells multiplied by cloning?

Answer 27

Modified bacterial cultures grown large scale in industrial fermenters. Conditions carefully monitored + controlled for optimum growth. Reproduce asexually by cell division. Genetically identical clones produced. Insulin gene expressed in bacteria so it produces insulin by protein synthesis. For human insulin, so produces human insulin. Culture solution drawn off at intervals + human proteins purified.

Question 28

What is the purpose of genetically modified crops?

Answer 28

Produce pest + herbicide resistant species, higher yield, improved ecological range (cope with drought/frost), improved food quality (taste, texture, shelf life, colours) + production of substances for medical/pharmaceutical use. Good for starving countries.

Question 29

What are 3 diff ways to deliver new genes into plant tissues for GMOs?

Answer 29

Ti plasmid, gene guns + plant viruses.
Gene of interest inserted along with fluorescent marker/antibiotic resistant gene so transformed cells identified + grown using tissue culture method.

Question 30

How is the Ti plasmid used to deliver new genes into plant tissues?

Answer 30

Ti plasmid found in bacterium A.tumifaciens, bacterial species that readily invades damaged plant tissue. Having invaded plant tissue, bacteria releases Ti plasmids into plant cells. By engineering gene of interest into Ti plasmid, plants have new gene spliced into own DNA + should start expressing protein.

Question 31

How are gene guns used to deliver new genes into plant tissues?

Answer 31

Tiny pellets coated with DNA, fired through cellulose cell walls of plant cells.

Question 32

How are plant viruses used to deliver new genes into plant tissues?

Answer 32

Many viruses that infect plants. Can be engineered to carry gene of interest into infected cell.

Question 33

What are the most commonly used methods to insert gene of interest into animal cells?

Answer 33

Viruses (same principle as GM plants), microinjection of DNA into fertilised egg, liposomes, electroporation.

Question 34

What are some of the techniques used to deliver functional form of gene into target tissue for gene therapy?

Answer 34

Retro + adeno viruses + liposomes. Recently, non human infective virus chosen to treat haemophilia B so had no immunity to virus + didn’t insert DNA into host cell chromosomes.

Question 35

Why was a non human infective gene chosen for gene therapy?

Answer 35

No immunity to virus as immune response would stop viruses from infecting cells + preventing gene delivery. Also not inserting host cell chromosome as this could cause cancer as insertion of functional gene disrupted other genes, causing uncontrolled cell division.

Question 36

Give a different application of gene therapy.

Answer 36

Used engineered cold sore virus to infect cancer cells. Gene tech used to stop it from replicating in normal cells, just cancer cells. Added genes to virus which made cancer cells more visible to immune system so detected as foreign + destroyed.

Question 37

How are DNA chips used?

Answer 37

DNA sampled + amplified using PCR. Digested into smaller fragments using restriction endonuclease enzymes. Made single stranded + label added. Fragments washed over chip + complementary sequences attach to probes. Signal from each well digitally recorded, more labels in 1 well = greater signal.

Question 38

Give a different application of DNA chips.

Answer 38

Identification of genes that are (not) expressed in cancer tissue. mRNA isolated from tissue + reverse transcriptase used to produce cDNA, label added. Labelled cDNAs exposed to DNA chip + signal strength recorded. Signals compared to healthy tissue + can determine whether genes up/down regulated. Use diff coloured label.

Question 39

What are the 4 possible outcomes from each well on DNA chip when identifying gene expression?

Answer 39

No sample cDNAs hybridises to probes on DNA chip, red + green emit same signal strength so no diff in gene expression, those giving off green (healthy) signal indicate genes switched on in healthy, off in cancer or red signals indicate genes operating in cancer but not in healthy. May be oncogenes.

Question 40

How is DNA microarray analysis used in clinics + individualised medicine?

Answer 40

Used to understand which drugs most effective for chemo + reduce side effects e.g. breast cancer. If cancer caused by HER2 gene, trastuzumab effective, if not, causes heart damage.
Individualised drug treatments based on genotype. Patients with certain genes don’t respond to certain treatments. e.g. codeine. -> ultra rapid metabolisers convert into morphine, could kill. Poor metabolisers wouldn’t respond at all.

Question 41

What have the genome projects allowed/helped?

Answer 41

Classification of organisms through creation of phylogenetic trees (can see how related organisms are, common ancestry), disease detection (microarrays, gene therapy), genetic risk factor detection (BRCA1 + 2), pathogen genomes sequence to understand infection (antibiotic resistance, track pandemic, more effective treatments), protein modelling + identification of people that respond correctly to chosen drug.

Question 42

What is the purpose of protein modelling?

Answer 42

By knowing gene sequence, can determine order of AAs in protein + predict secondary + tertiary structure which can be used to develop drugs (enzyme inhibitors, if shape of active site visualised, inhibitor can be designed to block it)

Question 43

What are some of the safety precautions for gene technology?

Answer 43

Bacteria strains poorly adapted to human physiology (slow growing, only grow at temps higher than human body temp), engineering suicide genes activated if pH/temp outside narrow limits + high containment levels (filters, careful monitoring of air in purpose built labs)

Question 44

How can a DNA probe be made radioactive?

Answer 44

Labelling 5’ end with 32P.

Question 45

Why must unhybridised DNA probes be washed off during gel electrophoresis?

Answer 45

Fluorescently labelled probes will still show up. Now no longer necessary but in past.

Question 46

In terms of the PCR, do the temps vary?

Answer 46

Yes, they depend on the type of DNA being amplified but are still higher/lower to be functional.

Question 47

What are some of the ethical issues related to GM crops?

Answer 47

Not natural, superweed creation as genes transferred to weeds, greater ecological range so outcompete non crop species, some insects resistant to toxic effects with Bt gene + compounds produced by them could cause allergies.

Question 48

Give 4 examples of transgenic organisms, their modification + consequence.

Answer 48

Chicken - miR-24 antibody obtained from eggs, treats skin cancer.
Cow - Gene for protein alpha-lactalbumin so milk more similar to human.
Sheep - Factor 8 purified from milk to treat haemophilia + serum proein alpha-1-antitrypsin to regulate protease activity + treat CF.
Goat - Anticoagulant anti-thrombin.

Question 49

How common is heterozygosity?

Answer 49

Quite rare.

Question 50

What are the 4 diff types of codeine metabolisers?

Answer 50

Poor, intermediate, extensive or ultra-rapid.

Question 51

What are some of the containment measures for GMOs?

Answer 51

Appropriately licensed + purpose built labs with effective air filters, good cleaning + access procedures, disinfectant procedures, poorly adapted bacterial strains which can’t survive in humans + use strains with suicide genes that are activated if conditions not within spec pH or temp limits.

Question 52

What are the ethical issues with genetic screening?

Answer 52

Abortion for Down’s/other diseases. Should insurance company/employer/life insurer know details?

Question 53

What are some of the ethical issues with gene therapy?

Answer 53

Expensive - better spent on reducing waiting lists/antibiotics + vaccines for 3rd world? Not without risk, could disrupt host DNA. In French gene therapy trial for SCID, 2/11 died from leukaemia. After this, all research stopped for 10 years.

Question 54

What are some of the other possibilities with gene technology?

Answer 54

Personalised medicine/pharmacogenetics + biobanks.

Question 55

How can DNA be extracted?

Answer 55

Add salt + cold water to plant, grind with pestle + mortar, strain off larger bits + mix with detergent to break down cell membranes, leave 5-10 mins. Add protease to separate DNA from chromosomes. Add layer of cold ethanol, DNA precipitates out as series of white strands.

Question 56

Why add salt to DNA for extraction?

Answer 56

Reduces solubility of DNA in water so easier to be precipitated out.

Question 57

Suggest a possible source of error with the use of the PCR in legal cases.

Answer 57

Contaminated sample + errors in replication process.