4.5 - Application of Reproduction and Genetics

define the human genome project

define the human genome project

The sequencing of the nucleotides in the human genome to identify all the genes present AND which chromosome each gene is on (gene loci)

Define Sanger sequencing

The original method used to sequence the human genome

Define electrophoresis

The method that separated the DNA fragments on the basis of their size that determined their migration rate through a gel

What non-coding sections of the chromosome (10%) were not investigated by the human genome project

The centromere (middle of the chromosome) and the telomeres (ends of chromosome)

define genetic profiling

The study of an individual’s unique non coding DNA, the STR’s

define STR

Short tandem repeats - sequences of bases in introns that repeat up to hundreds of times

define PCR

Polymerase chain reaction - uses the semi-conservative replication of DNA from samples to amplify the mass of DNA present without changing the sequence

Define primer

A strand of DNA that pairs to a longer DNA strand - enables DNA polymerase to attach for replication

Define probe

A short section of DNA that is labelled with a fluorescent (emits light) or radioactive marker - it is used to identify the position of the complementary base sequence

define restriction enzymes

Bacterial enzymes that cut the sugar phosphate DNA backbone at specific nucleotide sequences to cut DNA into many small fragments

define sticky ends

The unpaired bases that protrude from a length of DNA when a staggered cut is made by a restriction enzyme. The bases at either end are complementary (so would bind together or with a different strand cut with the same enzyme)

define introns

Non coding sections of DNA, removed post transcription so do not contribute to sequence of bases in mRNA

Define reverse transcriptase

An enzyme derived from a retrovirus - they catalyse the synthesis of DNA from RNA templates strands

define vector

A term to describe the virus of bacterial plasmid that can carry foreign genetic material into target cells

define plasmid

Small circular loop of double stranded DNA in bacterial cells - they are self replicating within the cells

define marker genes

Additional genes included in the transfer of genes into plasmids that can identify if desired gene has been taken up by the plasmids - antibiotic resistance is commonly used

define clone

Genetically identical cells formed from singles cells (or from parent organism)

define gene therapy

Treatment of a genetic disease by replacing the defective allele with an allele cloned from a healthy individual (and inserted into the genome)

Define germ line therapy

When the corrective gene is inserted into “germ line cells” (cells that will replicate to form the organism

Define somatic cell therapy

When the corrective gene is inserted into body cells in affected tissue - but the corrective gene is not inherited by daughter cells

define liposomes

Hollow phospholipid spheres that can be used to transfer molecules, such as genetic material, into cells

define stem cells

Unspecialised cells that can develop and differentiate into many different types of cell

define totipotent

Cells that retain the ability to differentiate into every cell type within an organism (e.g. embryonic cells)

benefits of the human genome project (7)

• help us understand diseases to improve their diagnosis and treatment

• Improve design of medication

• More accurately predict the effects of drugs

• Identify mutations linked to different forms of cancer

• Improve disease risk assessment

• Use in bioarcheology, anthropology and evolution

• Advance forensic applied sciences

Why are 2 different primers needed for PCR

Each end of the strand of DNA has a different nucleotide sequence

why is it important to use primers that are specific to a certain gene on each chromosome

Enables the gene to be amplified

why is it necessary to express the quantity of genes as a ratio

It does not matter how many cycles of PCR there are the ratio will stay the same

Limitations of the original human genome project (6)

• volunteers from Europe

• unknown age of donors

• unknown ethnicity of donors

• europeans can’t represent global genetic diversity

• unknown medical history of donors

• small sample size - limits genetic diversity of sample group

why 100k? (4)

• Sequence the DNA of 100,000 people in the uk = larger sample size

• not anonymous - informed consent

• able to collect age, ethnicity, + history of disease especially genetic disorders

• improved by increased global international involvement

aims of 100k (8)

• create an ethical and transparent programme based on consent

• bring benefit to patients and set up a genomic medicine service within the NHS

• kick start the development of the human genomics industry

• improve the accuracy of disease diagnoses → early diagnosis/screening

• better predict the action of drugs

• improve the design of drugs → drugs could be developed to target specific gene sequences - to “silence” the genes

• find new ways of treating genetic diseases

• explore the possibility of tailoring therapies to treat a disease in an individual person

issues with the 100k genome project (2)

• who owns the sequences? if a gene is identified could it not be used for commercial gain? but how could we develop the biotech medicine without commercial partners

• what can your DNA reveal about you? in the last 70 years people have been separated from their families as they displayed features that were atypical to their aligned ethnic group (apartheid) → could cause discrimination

concerns of the 100k genome project (3)

• an individual may want their genome mapped out for early diagnosis so early treatment or lifestyle decisions/counselling to prepare

• people may not want to know their genome as we would be living with the knowledge and insurance companies may misuse data/social stigma of disease

• increase chance of termination if foetal screening (rich have more access). embryo screening opens up a minefield of features that can and can’t be selected - yes we can avoid implanting embryos with genetic diseases - but could open up concerns linked to eugenics

steps of sanger sequencing (5)

1. of single strand DNA fragments (cut out using restriction enzymes)

2. PCR - amplification of fragments

3. complementary strands produced - but removed an OH from carbon 3 of terminal base pentose sugar

4. amplified (PCR) and terminal base labelled with a fluorescent marker - specific to each base!

5. used gel electrophoresis to separate by size and each band had marker to identify it’s terminal base

negative of sanger sequencing

too slow and had to build the genome fragment by fragment. 3 billion to code but 1 million a year. too expensive

benefits of next generation sequencing (4)

• more automated

• cheaper (£6,000 vs £6,000,000 for human if used now)

• faster

• increased accuracy

beyond human sequencing - why bother? (3)

• benefit of access to gene sequences: proving evolutionary links, use in breeding programmes to ensure genetic diversity

• for diseases e.g. malaria

  • kill the parasite or vector

  • mosquitos modified to produce antibodies against plasmodium

  • identify the gene that gives resistance to previous antimalaria

• silence the gene → difficulty = single vs multiple points

what is CRISPR (3 +1 points)

1. genome project has located the gene for drug resistance and identified the base sequence of the gene

2. knowing its sequence we can use the correct complementary RNA sequence to guide the Cas9 protein into position

3. the protein cuts the DNA and inserts a section of DNA to correct/silence the mutated gene

easier with single point than multiple mutations

ethical concerns of CRISPR (5)

• could lead to the editing of non target genes

• passed onto future generations

• irreversible alteration of the human gene pool

• contribute further to global health inequalities

• accessibility issues

what do non-coding introns contain

STRs (short tandem repeats) → the number of repeats varies between individuals and the number of repeats is heritable

process of the polymerase chain reaction (5)

1. sample of DNA mixed with: TAQ polymerase (automates the repetitive step of amplifying specific DNA sequences), nucleotides with all 4 bases (in excess), primers (short strand of complementary DNA that binds to one end of a template so polymerase can attach)

2. sample heated to 90ºC in a thermoclyer to unwind and separate strands

3. cooled to 55ºC → allows primers to bind to complementary sequences (anneal)

4. reheated to 75ºC → the extension/elongation phase as polymerase adds complementary nucleotides

5. and repeat

why do crime scene investigators wear protective clothing? (limitations of PCR)

contamination, original sample cross contaminated, sampler contamination, apparatus contamination, aur borne contamination

if mutations can occur during DNA replication - what can happen during PCR? (limitations of PCR)

errors due to mutations

why are mutations more problematic in PCR? (limitations of PCR)

during replication in cells, polymerase enzymes proofread and remove errors (TAQ polymerase does not)

why would the rate of production slow down (in a sealed reaction vessel) (limitations of PCR)

reagents will reduce in concentration - and repeated temperature changes will denature enzymes involved (NOTE: single strand molecules at stage 2 can reduce replications - but proportions will still be in same ratio)

Other chemicals… (limitations of PCR)

can inhibit process. plant based phenolic compounds, humic acids formed from degrading plant matter is present in old samples (bioarchaeology and the haem group binding to magnesium is needed for polymerase function)

explain gel electrophoresis (3 points)

• separate DNA according to fragment size. the fragment size, in this case will depend on the number of repeats at a range of given STR regions

• enzymes will cut at specific loci and the combined fragment size will be specific to an individual hence, they can be compared

• samples are added to the wells at the top and the gel is subjected to an electrical current

steps of gel electrophoresis (4)

1. restriction endonucleases are utilised to cut the samples of DNA or RNA into smaller fragments so they can be easily separated and examined

2. DNA transferred from agarose gel to an inert nylon mesh (no further migration)

3. probes → short strands of DNA that are complementary to the STRs, and have either radioactive or fluorescent markers attached to them are used - they bind to the STRs

4. mesh transferred to a film (either x ray or wavelength sensitive)

why is gel electrophoresis used (3)

• isolate, identify and characterise properties of DNA fragments into many different situations and at many different points during the cloning process

• separation of DNA fragments

• for genetic profiling to investigate crime scenes

pros of DNA profiling (5)

• non invasive

• tiny amounts needed

• has reversed wrongful convictions

• exonerates suspects

• provides a profile of humans

cons of DNA profiling (5)

• violation of civil privacy

• databases may be hacked and misused

• profiles are probabilities not certainties

• access must be highly regulated

• wrongful convictions - could plant DNA

why are introns more significant than exons for genetic profiling?

exons are coding (and they don’t differ between individuals). introns are non coding and they vary due to the different number of STRs present in each

why are restriction endonuclease enzymes called restriction endonuclease enzymes

restriction enzymes cut DNA at specific sites known as restriction sites (and they cut between a specific sequence) they result in sticky ends

key steps of genetic engineering (5)

• identify

• isolate

• insertion (into the genome of vector)

• transfer (the vector to a host cell)

• identify cells and clone

What is isolating the gene (using restriction endonuclease enzymes)

Directly cutting the identified gene from a DNA sequence

How is isolation of the gene done (using restriction endonuclease enzymes) + different types of ends

Restriction endonuclease isolated from bacteria to cut DNA into fragments to isolate target gene - staggered cut = sticky ends so palindrome if read back to back, straight ends = add sticky ends later

What happens if the restriction site is not exclusive to the end of the gene (isolating the gene using restriction endonuclease enzymes)

The target gene will be cut into fragments

What happens if the DNA is cut at the gene ends (isolating the gene using restriction endonuclease enzymes)

The isolated “gene” will contain the non coding introns that are normally spliced out during post transcriptional processing prior to mRNA forming. Bacteria don’t modify mRNA (no introns - so any protein translated by a bacteria will be non functional as wrong primary sequence) additional amino acids in the polypeptide chain

Steps for isolating the gene (using reverse transcriptase) (4)

1. Enzymes extracted from retrovirus (they reverse transcription)

2. So form DNA from a mRNA sequence isolated (no introns - coding sequences only) → benefit = mRNA can be easier to isolate as large quantities in active cells with specific functions e.g. ß-cells in pancreas that produce insulin

3. DNA referred to as copy DNA (so cDNA) differs to original DNA (as no introns) → but it is single stranded so……..

4. DNA polymerase required to form double strands prior to insertion into vector

Steps of recombinant DNA (6)

1. Prepare bacteria

   1. Destabilise cells walls (EDTA)

   2. Dissolve the cell membrane with detergents

   3. Denature membrane proteins

   4. Separate the plasmids (centrifuge)

2. Same restriction enzymes that cut out gene used to cut open plasmid

   1. So there is complementary sticky ends

3. DNA ligase is used to join gene and plasmid

4. Plasmid is now recombinant DNA

5. Bacteria take up recombinant plasmid

6. Clone in fermenter (large numbers rapidly)

Steps for genetic markers (6)

1. 2 antibiotic resistance markers (e.g. amp and tetra) added to bacteria

2. Gene (e.g. Insulin) added in a break in antibiotic resistance marker to tetra

3. Make master plate of bacteria

4. Print onto plate with amp virus

5. Print onto plate with tetra virus

6. Bacteria that has resistance to amp but not tetra has the insulin gene

Pros for genetic engineering in bacteria (5)

• rapid large scale production of human proteins without ethical objections animal use e.g. insulin and human growth hormone

• Lower long term risks to human health (no human contamination of products)

• Environmental benefits → bacteria absorbing heavy metals from waste etc.

• Tooth decay prevention → modified bacteria outcompete acid producing naturally occurring bacteria vaccine production

• Agricultural pest management → toxic effect on pest species

Cons of genetic engineering in bacteria (6)

• modified plasmids can transfer easily between bacteria → antibiotic resistance could be shared with pathogenic species

• Inclusion of non target genes such as oncogenes or protoncogene “triggers”

• Long term risk of modified bacteria causing environmental triggers

• Potential long term effect of tooth decay prevention

• Contamination of products (non target genes)

• Long term risk of modified bacteria causing environmental consequences → toxic effect restricted to pathogens?

Steps of a gene gun (4)

1. Gold or tungsten pellets containing a preparation of plant genes (inert)

2. Penetrate membrane of cultured cells

3. Tissue culture

4. “Explants” extracted and grown on (hormone treatment required to get cells in tissue culture to differentiate)

What is electroporation

An electric charge is applied to cells, which permeabilises the membrane so that DNA can enter the cell

What is microinjection

A fine needle is used to directly inject the recombinant DNA into a single host cell

What is Agrobacterium tumifaciens

A soil dwelling gram negative bacterium. In “nature” infects plants and has a DNA sequence that inserts itself into the plant’s chromosome (tDNA → induces plant tumour growth - form galls on plants. SO this organism has the ability to transfer its genetic material into plant cells)

Steps of GM plants and Agrobacterium tumifaciens (6)

1. Extract bacterial plasmid

   1. Cut plasmid within the tDNA restriction enzyme (sticky ends)

2. Cut the desired gene from other species (same restriction enzyme)

3. Target gene inserted into bacterial plasmid with use of DNA ligase

4. Plasmid taken up by bacteria reproduction etc.

5. Bacterium becomes a vector to introduce the tDNA into the plant cells

6. New plant cells grown in tissue culture - and plants cloned from “explants”

What is bacillus thuringiensis (BT tomatoes)

A free living soil bacteria → has a plasmid gene that produces an insecticide protein

How are tomato plants modified (BT tomatoes)

GM bacteria using Agrobacterium tumifaciens to modify the tomato plants to produce the insecticide BUT! Has to be only transcribed and translated in leaf cells or chemical in fruits impact flavour

Benefits of BT tomatoes (1)

Decreased reliance on pesticides (cost/environmental → non target species not harmed)

Benefits of tomatoes ANtisense (2)

Longer shelf life, reduced wastage

What happens to a normal tomato over time (4 steps)

1. Polygalacturonase gene is transcribed → mRNA

2. Translated to produce PG

3. PG breaks Pecitin cell walls down

4. Soften/over ripe and decomposes

What happens to a tomato over time when it has been genetically modified using A.tumifaciens (4 steps)

1. Introduce second copy of PG g1 gene but reversed so “ANTISENSE”

2. So when transcribed 2 copies of complementary mRNA produced

3. mRNA strands bind together

4. No translation so no PG enzyme to break pectin down

How does conventional sugar beet react when treated with herbicides

Herbicides can increase productivity by killing competing weed species but they can reduce/delay growth of crop species or can only be used before planting the crop

How does roundup ready sugar beet react when treated with glyphosphate herbicides

• Herbicides can increase productivity by killing competing weed species

• Plants contain a gene to give resistance

• We can spray herbicides anytime without harming our crop

Pros for genetic modification of crops (5)

• Gm crops could be utilised to produce pharmaceutical chemicals such as antibodies, hormones, vaccines, (PHARMING)

• If crops are resistant to pests we can reduce reliance on pesticides

• Food could be enhanced to improve nutritional value → golden rice produces vit. A to reduce blindness in developing nations

• Increases crop yield due to reduced loss from pests or increased tolerance to draught

• Crops can be modified to become resistant to herbicides → weed killer chemicals can be applied at any time to kill weeds (reduced competition for crop species to increase yield)

Cons for genetic modification of crops (7)

• Pollen from GM crops transfer to wild species (if similar). Could reduce our ability to control weed species or disrupt food chains

• GM crops could “fertilise” organic crops - compromising the industry

• Potentially new proteins would be present in our crop species - possible unknown side effects of eating the GM crop

• A limited number of wealthy biotech companies could result in reduced crop diversity, reduced biodiversity and gene pool and loss of potentially useful genes

• Pest resistant crops could result in pest species developing resistance to the “gene” protein expressed in the GM

• Who owns? Who can develop? Increased expense to food producers as seeds for GM crops have to be grown under license

• The marker genes used in the process that give antibiotic resistance could transfer to the bacteria in human intestines - increased resistance in pathogens!

Genetic screening has the potential to……. (4)

• Facilitate the selection of a potential treatment for a condition

• Diagnose a disease by identifying the presence of an allele

• Enable families using IVF to select embryos without the causative genes so they can avoid having children with genetic diseases

• Provide tailored medical advice to those identified as being at risk from preventable diseases

What can carrier screening do

• Identify if unaffected individuals “carry” recessive alleles for genetic diseases

• Inform decision on having children or antenatal screening to check if child will be a “sufferer” and have 2 copies of an allele

Who is carrier screening offered to

Those with family history of a disease

What may carrier screening lead to an increase in

Pregnancy terminations

Issue with pre natal screening (1)

Commercialised testing preying on paranoia and is completely unregulated

Benefit of post natal screening (1)

Early diagnosis so prompt treatment/preventative measures

What is pre symptomatic screening

Genetic testing performed in an individual who does not show symptoms of the disorder but who is at risk of developing the disorder

What does pre symptomatic screening allow individuals to do

Make informed lifestyle choices, reduced exposure to “triggers”

Other uses of genetic screening (3)

• confirmation of disease following symptom identification → reduces time before treatment

• Confirm family links

• Screen individuals/volunteers (forensic)

Issues with genetic screening (1 with 3 subpoints)

Commercial testing kits are:

• Unregulated - so can exploit fears

• Only probabilities

• Positives can lead to unfounded patient anxiety

Step 1 of gene therapy

A viral vector has the gene inserted

Step 2 of gene therapy

The gene is transferred to the target cell

Step 3 of gene therapy

Gene expressed in target cells (if taken up)

What else can be used for gene therapy (other than a virus)

Plasmids and naked DNA

What is somatic gene therapy (4 points)

• corrective gene inserted into target cells in affected tissues

• Provides therapy → relief from symptoms

• Gene not inherited by daughter cells

• Treatment requires repeating

What is germ line gene therapy

• corrective gene inserted into germ line cells

• I.e. oocyte → a cell that will result in the organism and therefore the gene passed onto all subsequent cells

Issues with gene therapy (3)

• unpredictable nature of modifying genes

• Interaction of genes → potential to activate or silence other sequences

• Unknown long term potential effects

What gender could get DMD

Men

What does DMD do (3 points)

1. A deletion in an exon

2. Reading of transcribed RNA is blocked

3. No dystrophin produced (which is a structural molecule in muscle protein) → muscle wastage and so limited life expectancy

Therapy for DMD (2 points)

1. Antisense oglionucleotide that is complementary to faulty exon binds to mRNA so it is double stranded preventing translation

2. A shorter but viable form of dystrophin produced

What is cystic fibrosis and what does it do (7 points)

• homozygous recessive

• Over production of mucus

• CFTR → cystic fibrosis trans membrane not coded for

• So no transport out of cells of chloride ions

• So no sodium ions follow

• So less water moves out by osmosis

• So mucus thick and sticky

Applying GM to treat cystic fibrosis (4 points)

1. Gene coding for normal CFTR identified, isolated and replicated

2. Gene coding for CFTR inserted into liposomes - phospholipid spheres

3. Liposome fuses with cells’ membranes and release genes into cells

4. Normal gene expressed during translation

Limitations of applying GM to treat cystic fibrosis (3)

• may not insert into all target cells

• Could insert gene into non target cells

• Treatment only → repeat needed as cells will die and not pass gene on