Genetics - 3.5 Genetic Modification and Biotechnology

Question 1

Understandings:

Answer 1

1. PCR can be used to amplify small amounts of DNA
2. Gel electrophoresis is used to separate proteins or fragments of DNA according to size
3. DNA profiling involves comparison of DNA
4. Genetic modification is carried out by gene transfer between species
5. Clones are groups of genetically identical organisms, derived from a single original parent cell
6. Many plant species and some animal species have natural methods of cloning
7. Animals can be cloned at the embryo stage by breaking up the embryo into more than one group of cells
8. Methods have been developed for cloning adult animals using differentiated cells

Question 2

Gene technolodies (6)

Answer 2

1. DNA sequencing
2. DNA profiling
3. Genetic screening
4. Genetically modifying organisms
5. Gene therapy
6. Cloning

Question 3

DNA sequencing

Answer 3

• finding the order of the bases in the genome (eg. the Human Genome Project)

Question 4

DNA profiling (fingerprinting)

Answer 4

• where a unique pattern is produced for each individual based on the variations in each person’s DNA

Within the non-coding regions of an individual’s genome there exists satellite DNA – long stretches of DNA made up of repeating elements called short tandem repeats (STRs)
As individuals will likely have different numbers of repeats at a given satellite DNA locus, they will generate unique DNA profiles

Probe = a single strand of DNA that will stick to a gene of interest (it is made of a length of DNA that is complementary to the gene of interest) - it is usually made with radioactive or fluorescent tags (if the probe is washed over the gel then the presence of that gene will be shows

If radioactive tags are used = an autoradiograph (autorad) is created

Microsatellites = repeated sequences that vary from about 16 to 60 bases long (if duplicated thousands of times = forms stretches of DNA) - these regions = VNTR (variable number of tandem repeats (different for each individual in both # of base paris in the repeat section and # of times it is repeated)) (in comparison, STR = Short tandem repeats)

Method of DNA profiling:
1. DNA is extracted from the sample
2. The DNA sample is treated with the restriction enzyme Hinfl which cuts the DNA in several places
3. Gel electrophoresis allows different lengths of DNA to be separated
4. Treatment of the gel with radioactive probes complementary to the part of the sequence of the VNTR or STR regions to be analysed
5. Autoradiograph is produced

The procedure involved is common for both:

A DNA sample is collected (e.g. from blood, semen, saliva, etc.) and then amplified using PCR
Satellite DNA (with STR sequences) are cut with specific restriction enzymes to generate fragments
Fragment length will differ between individuals due to the variable length of their short tandem repeats
The fragments are separated using gel electrophoresis and the resulting profiles are compared

Uses of DNA profiling:
1. forensics - to match suspect with crime scene evidence (micro-satellite DNA = analysed to produce a DNA profile (fingerprint) then used to compare DNA samples)
2. paternity testig
3. identifying the presence of a particular gene in a family (eg cystic fibrosis)
4. Genetic relatedness of different organisms
5. Breeding programmes (eg, checking that captive populations of endangered species are not inbred)

Question 5

Genetic screening

Answer 5

• testing an individual for the presence or absence of a gene (eg. cystic fibrosis)

Question 6

Genetically modifying organisms (GMOs) (animals, plants and bacteria)

Answer 6

• adding DNA from other organisms or by silencing unwanted genes

GMOs can be created by modifying their DNA in 3 different ways:
1. Adding a foreign gene
(a foreign gene is added which will enable the GMO to carry out a new genetic program - organisms altered this way = TRANSGENIC)

2. Altering an existing gene
   (existing gene present in organism may to altered to make it express at a higher level or in a different way (also used for gene therapy))
3. deleting/’turning off’ a gene
   (an existing gene may be deleted/deactivated to prevent the expression of a trait)

GMO debate: https://ib.bioninja.com.au/standard-level/topic-3-genetics/35-genetic-modification-and/gmo-debate.html

Question 7

Gene therapy

Answer 7

• defective genes are replaced in the diseased individual (eg. cystic fibrosis)

Question 8

Cloning

Answer 8

• identical organism is artificial derived from a single parent cell

Clones can occur naturally - via asexual breeding in plants/lower animals/identical twins (eg. bacteria and fungi - usually via binary fission/budding)
Natural methods of cloning in plants = cuttings/bulbs/cut-offs
Natural methods of cloning in animals = eg. starfish, annelids and platyhelminths by fragmentation.
(natural cloning = https://ib.bioninja.com.au/standard-level/topic-3-genetics/35-genetic-modification-and/natural-cloning.html)

Artificial cloning:
https://ib.bioninja.com.au/standard-level/topic-3-genetics/35-genetic-modification-and/artificial-cloning.html

(((Cloning animals by somatic nuclear transfer = on another flashcard)))

Question 9

Techniques / tools commonly used in gene technologies (5)

Answer 9

1. DNA extraction
2. Restriction enzymes
3. Gel electrophoresis
4. Polymerase Chain Reaction
5. Plasmid Vectors

Question 10

DNA extraction

Answer 10

• DNA is removed from the individual’s cells nucleus so that it can be prepared for analysis/transfer/modification (eg. GMOs, DNA profiling, DNA sequencing) (eg. blood sample)

Steps:
1. isolate the SNA from the rest of the cell
(mechanically breaking cells open -> use detergents and enzymes to break down cell walls and membranes)
2. remove the unwanted cell debris
(either by filtering the extract or centrifuging the mixture - the filtrate will now contain the wanted DNA (+ some unwanted proteins))
3. remove unwanted proteins
(adding a protease enzyme = destroys proteins (some labs = add phenol))
4. precipitate out the DNA
(done my pouring a layer of ice-cold ethanol over the surface of the filtrate

Question 11

Restriction enzymes (endonuclease)

Answer 11

• used to ‘cut’ DNA for analysis / so that the desired genes can be removed (eg. GMOs, DNA profiling) (eg. bacteria attacked by a virus - cut up the invading viral DNA) (over 400 discovered - many scientists have harnessed this ability and used these enzymes as DNA scissors)

(they are names according to the bacteria species they were first isolated from, followed by a # to distinguish diff enzymes isolated from the same organism)

= restriction enzymes make a cut = a blunt end and/or a sticky end
= these ends can be glued to some DNA from a different source that has been cut by the same restriction enzyme (= ANNEALING)

Question 12

Gel electrophoresis - separating DNA fragments

Answer 12

• separates lengths of DNA based on size = a ‘picture’ can be made (eg. DNA profiling)

Process depends ont he following facts:
1. when the restriction enzymes have cut DNA, the fragments will be of different lengths
2. the DNA is strongly negative due tot he phosphates in the nucleotide

Method:
1. AGAROSE GEL (transparent - separates DNA thing based on size) is made with wells in it
2. Whole gel box is flooded with a buffer solution which will allow electricity to pass through (strong neg charge) = “porrus”
3. prepared DNA is carefully introduced into the small wells
4. The electrodes are turn on - at each end of the gel box - with the positive electrode the furtherest away from the wells
5. DNA will move through gel = smallest pieces moving fastest towards pos electrode
6. The rate at which these fragments is inversely proportional to their length, so the exact size of the fragments can be calculated (eg. 25 kilo-bases long)
(simplest way to visualise fragments = dye them with a dye that binds to the DNA

https://ib.bioninja.com.au/standard-level/topic-3-genetics/35-genetic-modification-and/gel-electrophoresis.html

Purpose of this:
- if looking for a particular sequence -> gene is incubated -> sequence (complementary DNA) can be found = seperation technique

Question 13

Polymerase Chain Reaction (PCR)

Answer 13

• used to make many copies of DNA (ie amplify minute quantities of nucleic acid) (eg. DNA profiling, GMOs) = DNA amplification

Process:
(carefully chosen primer can latch onto a specific gene & make many copies)
1. Denaturing (95 deg,cels - DNA strands separate)
2. Annealing (55 deg,cels - primers bind template - DNA primers attach to the 3’ ends of the target sequence)
3. Extension (72 deg,cels - synthesise new strand) - polymerase added to make lengths
cycle repeats, A heat-tolerant DNA polymerase (Taq) binds to the primer and copies the strand

CONCERN = scientist must be careful to eep specimens clear of any contamination!

Question 14

Plasmid vectors

Answer 14

• … from bacteria are used to transfer genes from one organism to another (eg GMOs)

Question 15

Restriction enzyme cuts the double-stranded DNA molecule at its specific recognition site (eg:)

Answer 15

1. EcoRI from the bacteria Escherichia coli cut DNA at the recofnition site G/AATTC
2. BamHI from the bacteria Bacillus amyloliquefaciens cuts DNA at the recognition site G/GATCC
3. HindIII from the bacteria Haemophilus influenzae cuts DNA at the recognition site A/AGCTT
4. Taql from the bacteria Thermus aquaticus cuts DNA at the recognition site TC/GA

Question 16

“blunt end” - restriction enzymes

Answer 16

here DNA is cut straight across

Question 17

“sticky end” - restriction enzymes

Answer 17

= palindromic cuts
= they make staggered cuts giving sequences in which both strands read the same in the 5’ to 3’ direction

Question 18

ANNEALING =

Answer 18

these ends (a blunt end and/or a sticky end) can be glued to some DNA from a different source that has been cut by the same restriction enzyme

Question 19

Ligase enzymes - restriction enzymes -

Answer 19

(needed as you can’t just plonk complimentary bases in)

Combining the sticky ends of 2 diff. DNA strands is only temporary because only a few H bonds hold the ends together - the joins can be made permanent by DNA ligase enzymes (the enzymes that catalyse the formation of bonds between the phosphates and the sugars on the side of the DNA ladder during DNA replication and repair)

Question 20

Application of the PCR method:

Answer 20

1. police (when only have little bit of tisse = multiple = easier to identify criminals and eliminate the innocent from their inquires = more tests can be run)
2. Anthropologists/archaeologist = use to check ancient fossils
3. scientists = used to see if someone has a specific gene (eg. for cystic fibrosis) (by a careful choice of the primer, this can latch onto a specific gene and make many copies, then, by gel electrophoresis, gene can be identified
4. used to identify viral genes much quicker than normal methods and identify them earlier such as HIV
5. used to identify rapidly any prenatal genetic disorder from a few fetal cells

Question 21

how is it (DNA profiling) used in forensics

Answer 21

• left over blood tissues are analysed for their micro-satellite DNA (repeat sequences that vary from 16-60 bases long) which are then compared to existing DNA samples (Ie allowing for a conclusion if they are related or not to be concluded) - DNA is extracted from a sample. PCR is used to make copies of an area of a gen (STR/VNTR) DNA is cut up with restriction enzymes, separated by electrophoresis incubated with a DNA probe (with a tag

Question 22

Probe (+ autoradiograph) =

Answer 22

a single strand of DNA that will stick to a gene of interest (it is made of a length of DNA that is complementary to the gene of interest) - it is usually made with radioactive or fluorescent tags (if the probe is washed over the gel then the presence of that gene will be shows

If radioactive tags are used = an autoradiograph (autorad) is created

Question 23

Microsatellites =

Answer 23

repeated sequences that vary from about 16 to 60 bases long (if duplicated thousands of times = forms stretches of DNA) - these regions = VNTR (variable number of tandem repeats (different for each individual in both # of base paris in the repeat section and # of times it is repeated)) (in comparison, STR = Short tandem repeats)

Question 24

Genetically modified bacteria / other micro-organisms

Answer 24

eg,
1. manufacture of human insulin/growth hormone by transgenic bacteria
(application: gene transfer to bacteria using plasmids makes use of restriction endonuclease and DNA ligase - production of human insulin in bacteria as an example of the universality of the genetic code allowing gene transfer between species)
2. yeast cells modified to make hepatitis B vaccine
3. Bacteria modified to ‘clean up’ oil spills

(Bacteria contains circular DNA = PLASMIDS)

(Simple) method of modifying bacteria =
1. isolate a gene of interest using restriction enzymes
2. remove plasmids from bacteria
3. cut plasmid with the same restriction enzyme
4. stick cut plasmid and gene of interest together using ligase enzyme
5. new recombinant plasmid inserted back into bacteria
6. bacteria reproduce asexually and express insulin gene

Question 25

Genetically modified crops (examples + benefits/risks) - know one eg really well!!! (ie golden rice)

Answer 25

genetic modification of plants = gene transfer by bacterial vectors/other methods = allowing genes to be inserted into plant DNA (plants can also reproduce asexually)

eg.
1. golden rice (with Vit. A (beta-carotene))
2. nitrogen fixation by non-legumes
3. (more on pg 15 of booklet)

Golden rice:
= rice contains beta-carotene = when ingested by humans = human body converts it to vit A
Benefits:
- starving people = easily get vit A in rice + allows general people to get more than the necessary amount of vit A (= avoiding serious medical conditions (eg blindness))
Harms:
- none (eg. also used in carrots), maybe: disrupt local ecosystems if an ecologically limiting nutrient or protein is produced

Question 26

Bt Crops and Monarch Butterflies

Answer 26

Refer to pages 16-17 of booklet for full explanation

conclusion from experiments/research:
“over 4 days 100% of the monarch larvae survived when eating milkweed plants not dusted with any pollen. The same occurred for those eating milkweed plants dusted with non-GM pollen. By day 4 only roughly 50% of the monarch larvae that ate milkweed plants dusted with Bt Pollen survived - with mortality rate decreasing just 1 day after consumption (ie they were eating less AND dying more)”

BUT another study found “there was no signif. increase in mortality with monarch larve were placed in or near an actual Bt corn field…”

Question 27

Genetically Modified Animals (bigggg ethics debate!!)

Answer 27

Examples:
1. growth hormone gene into salmon
2. knock out mice used to test cancer drugs
3. increased wool production in sheep

Basic outline:
Genetic modification usually involved the microinjection of the gene of interest into an already fertilised egg with is then implanted into a surrogate mother
ie.
1. two eggs are removed from a female + fertilised in a test tube

2a. one fertilised egg is left unaltered
3a. normal egg - cultured into an embryo - implanted into surrogate mother

2b. micropipettes inject (eg) rat growth hormone gene into fertilized egg
3b. transformed egg - cultured into an embryo - implanted into surrogate mother

4. two mice are siblings but one is 2x the size of the other

Question 28

Cloning animals by somatic nuclear transfer (eg dolly - 1996)

Answer 28

Process:
1. egg is removed from animal (1)
2. nucleus is removed
3. fuse cell (from another donner (animal (2)) and enucleated egg with electricity
4. egg fused with cell (cell division begins) -> embryo
5. implant embryo into animal (3)
6. clone born (clone of animal (2))

animal 1 = egg
animal 2 = cell
animal 3 = surrogate

Eg Dolly - died at 6 due to lung cancer and severe arthritis - she was born with a genetic age of the animal that the egg was originally taken from

=========================

Somatic cell nuclear transfer is a method by which cloned embryos can be produced using differentiated adult cells:

1. Somatic cells are removed from the adult donor and cultured (these cells are diploid and contain the entire genome)
2. An unfertilised egg is removed from a female adult and its haploid nucleus is removed to produce an enucleated egg cell
3. The enucleated egg cell is fused with the nucleus from the adult donor to make a diploid egg cell (with the donor’s DNA)
4. An electric current is then delivered to stimulate the egg to divide and develop into an embryo
5. The embryo is then implanted into the uterus of a surrogate and will develop into a genetic clone of the adult donor

https://ib.bioninja.com.au/standard-level/topic-3-genetics/35-genetic-modification-and/artificial-cloning.html

Question 29

Process of gene transfer (4 steps)

Answer 29

1. Isolation of gene and vector (by PCR)
2. Digestion of gene and vector (by restriction endonuclease)
3. Ligation of gene and vector (by DNA ligase)
4. Selection and expression of transgenic construct

Question 30

Gene transfer - Step 1: Isolating gene and vector

Answer 30

DNA can be isolated from cells by centrifugation – whereby heavier components such as nuclei are separated

The gene of interest can then be specifically amplified via the polymerase chain reaction (PCR)

Gene sequences can also be generated from mRNA using reverse transcriptase – these DNA sequences (cDNA) lack introns

A vector is a DNA molecule that is used as a vehicle to carry the gene of interest into a foreign cell

Bacterial plasmids are commonly used as vectors because they are capable of autonomous self-replication and expression

These plasmids may be modified for further functionality (e.g. selection markers, reporter genes, inducible expression promoters)

Other types of vectors include modified viruses and artificial chromosomes

Question 31

gene transfer - Step 2: Digestion with Restriction Enzymes

Answer 31

In order to incorporate a gene of interest into a vector, both must be cut with restriction enzymes at specific recognition sites

Restriction enzymes cleave the sugar-phosphate backbone to generate blunt ends or sticky ends (complementary overhangs)

Scientists will often cleave the vector and gene with two different ‘sticky end’ restriction endonucleases (double digestion) to ensure the gene is inserted in the correct orientation and to prevent the vector from re-annealing without the desired insert

Question 32

gene transfer - Step 3: Ligation of Vector and Insert

Answer 32

The gene of interest is inserted into a plasmid vector that has been cut with the same restriction endonucleases

This occurs because the sticky ends of the gene and vector overlap via complementary base pairing

The gene and vector are then spliced together by the enzyme DNA ligase to form a recombinant construct

DNA ligase joins the vector and gene by fusing their sugar-phosphate backbones together with a covalent phosphodiester bond

Question 33

Gene transfer - Step 4: Selection and Expression

Answer 33

The recombinant construct (including the gene of interest) is finally introduced into an appropriate host cell or organism

This process can be achieved in a variety of ways and is called transfection (for eukaryotes) or transformation (for prokaryotes)

Antibiotic selection is commonly used in order to identify which cells have successfully incorporated the recombinant construct

The plasmid vector contains an antibiotic resistance gene, so only transgenic cells will grow in the presence of antibiotic

Transgenic cells, once isolated and purified, will hopefully begin expressing the desired trait encoded by the gene of interest

Question 34

Comparison of Reproductive and Therapeutic Cloning

Answer 34

https://ib.bioninja.com.au/standard-level/topic-3-genetics/35-genetic-modification-and/artificial-cloning.html

Reproductive cloning: If the embryo is implanted into the uterus of a surrogate, a new cloned organism will develop

Therapeutic cloning: Embryonic cells can be induced to differentiate to create specific tissues or organs for transplantation