Application of reproduction and genes

Human genome project

designed to improve

Designed to improve the knowledge and understanding of genetic disorders and consequently improve their diagnosis and treatment

the project used method of sequencing data called “Sanger sequencing”/ chain termination method

The aims of the Human Genome Project

rlated technolgies

• Identify all the genes in the human genome and identify which chromosome each is on

• determine the sequence of base pairs in human DNA and store this information in databases

• improve tools for data analysis

• transfer related technologies to the private sector, to develop medical innovation

• address ethical, legal and social issues that may arise from the project

Findings of the Human Genome Project

• Humans have about 20,500, genes far fewer than anticipated

• there are more repeated segments of DNA that had previously been suspected

• fewer than 7% of the families of proteins were specific to vertebrates, emphasizing the close relationship between all living organisms

Electrophoresis

A lab technique that separates molecules on the basis of size, by their rate of migration under an applied voltage

Sanger sequencing

DNA is copied many times, making fragments at different lengths up to about 900 bases long

A fluorescent chain terminator nucleotide marks the end of each fragment, with four different colors for each base

Fragments are loaded onto a gel electrophoresis plate

smaller fragments move faster than larger and arrange themselves in size

Next Generation sequencing (NGS)

Can sequence an entire genome in a few hours because unlike sanger sequencing, they:

• are small scale- reactions can be done on a chip using small volumes of reactants

• use shorter fragments- up to 700 nucleotides

• run in parallel

• fast- because many reactions are done at

100k genome project

Uses Next Generation sequencing to sequence 100000 genomes from patients with cancer or rare disease and members of their family

Aims of 100k genome project

• Create an ethical, transparent program based on consent

• set up a genomic service for the NHS to benefit patients

• enable medical and scientific discovery

• develop a UK genomics industry

Moral and ethical concerns of the Human and 100k Genome Project

ownership of genetic information

• Ownership of genetic information: to ensure information is not misused and safeguards must be put in place for that individual. Eg.

  1. If a person is identified a disease, this information should not be used to set the insurance premiums or to deny them life or health insurance

  2. if DNA sequence suggests a particular ancestry, they should not be a pre-text for social discrimination

  3. no company should make financial profit from using a DNA sequence without permission

Moral and ethical concerns of the human and 100k Genome Project

identification of alleles sequences

genetic counselling

• The identification of alleles sequences: a patients DNA can be scanned for mutated sequences that may be correlated with future health problems. Some people don't want to have this knowledge but if the same health problems could affect relatives, it must be clear whether or not they have the right to the information

• genetic screening can be useful in with genetic counseling. If a family has a history of genetic defects, family members can consult a genetic counselor. Advice may be based on who has the condition, whether parents are closely related and frequency of mutated gene in the population. If a DNA sequence is available the genetic counselors, advice has more significance

Moral and ethical concerns of the human and 100k genome projects

embryos

Made during the process in vitro fertilization can be screened for the presence of alleles leading to conditions like cystic fibrosis Huntington's disease etc

a choice can then be made to only implant a healthy embryo

Moral and ethical concerns

embryo screening

Potential choosing of specific alleles to ensure specific characteristics

considered desirable characteristics

Moron ethical concerns

parents may wish

For the children to be screened to know if they carry sequences that might pre dispose them to adult diseases like Alzheimer's disease, cancer

a decision must be whether or not to tell the child the result

Society must decide if a parent has the right to such an knowledge about the child suggesting the child's DNA is a property of the parent

Moral and ethical concerns

storage and security

Of genomic data is a concern because of the potential for computer storage to be hacked

Examination of genomes of closely related organisms

classification

Allows interferences to be drawn concerning evolutionary relationships

provides a true phylogenetic classification and are compared with schemes based on phenotypic characteristics

Challenge of malaria

Common in sub-saharan Africa, Asia and Latin America

chemicals have been used to attack both the vector the mosquito- anopheles gambiae

and the parasite that causes the illness plasmodium falciparum

Killing the vector- Anopheles gambiae

Insecticides are used in indoor sprays to kill mosquitoes in buildings where they rest on walls after feeding on blood

mosquitoes have become increasingly resistant to insecticides

if a mosquito becomes resistant to an insecticide it becomes resistant to all insecticides

DNA sequence of the vector

Sequence is used to try and develop chemicals that can prevent the mosquito from transmitting malaria

by making it susceptible to insecticides

CRISPR-Cas9

Genetically modified mosquito produced using Gene editing technology

allows genes to be written into a genome

mosquito eggs were modified with the addition of a gene, that would allow them to synthesize an antibody against plasmodium

if the mosquito acquires plasmodium when taking blood from an infected person

the plasmodium would not survive in the mosquito the mosquito cannot spread infection

Killing the parasites- plasmodium falciparam

quinine

Disrupt plasmodium digestion of hemoglobin in the red blood cells

a toxic derivative of heamoglobin accumulates and kills the plasmodium

Killing the parasite

chloroquine

Disrupt digestion of hemoglobin in plasmodium's food vacuole

mutant plasmodium expels chloroquine from its food vacuoles faster than normal plasmodium

so there is not enough time for the drugs to have an effect

the mutant is consequently resistent to the drug

Killing the parasite

atovaquone

Kills plasmodium by acting on the electron transport chain in its mitochondria

resistance to it develops very rapidly

caused by single point mutation in the gene for cytochrome b

Killing the parasite

Artemisinin

Used in combination with other drugs

it also acts on the plasmodium in the red blood cells

resistance has now been detected

Genetic fingerprinting

A person's DNA profile is in their genetic fingerprint

represents only non-coding portions of DNA by two techniques:

• the polymerase chain reaction to make large numbers of copies of DNA fragments

• gel electrophoresis to separate the DNA fragments based on their size

Polymerase chain reaction PCR

Semi-conservative replication of DNA in a test tube

amplifies the DNA, makes it useful with very small or degraded smaple

DNA sample is dissolved in the buffer and mixed with

• Taq polymerase, this is a DNA polymerase from bacterium thermus aquaticus. Lives in hot springs and hydrothermal vents optimum temperature of 80 degrees but can remain active

• nucleotides containing four DNA bases

• short single stranded pieces of DNA called primers. Complimentary to the start of the DNA strand and bind to it, signaling Taq polymerase to start replication

technique depends on rapid temperature change

happens in thermocycler

Primer

The strand of DNA that base pairs with the end of another longer strand

making a double stranded section, to which DNA polymerase may attach prior to replication

Stages of polymerase Chain Reaction

• original target

• solution is cooled

• solution is heated

• sequence repeated

• Original “target” DNA is heated to 95 degrees for 30 seconds, separating into two single strands

• solution is cooled to 55 degrees for 20 seconds, which is cool enough for the primers to anneal to the complementary base sequences, on each of the single strands of DNA

• solution is heated to 70 degrees for 1 minute and taq polymerase catalyzes the synthesis of the complementary strand by adding complementary nucleotides and catalyzing the formation of phosphodieaster bonds in the phosphate sugar backbone. Elongation/extension phase

• sequence is repeated many times

Elongation/ extension phase

for each

For each initial fragment of double Shonda DNA

two identical double strands are produced

Limitations of polymerase Chain Reaction

contamination

Any DNA that enters the system by accident can be amplified

the contaminating DNA may be airborne

come from the experimenter or from contaminated reagents

Limitations of polymerase Chain Reaction

error rate

All DNA penemeries sometimes insert a nucleotide containing the wrong base

taq polymerase can't proofread or correct the errors

Limitations of polymerase Chain Reaction

DNA fragment size

most effciefent

Polymerase Chain Reaction is most efficient for making DNA with long base pairs, because taq polymerase can't correct errors

if a lower temperature, higher PH and a proofreading polymerase in addition to taq polymerase is used, a length of longer base pairs is generated

Limitations of polymerase chain reaction

sensitivity to inhibitors

Molecules in the sample may act as inhibitors and polymerase chain reaction is very sensitive to them

Limitations of polymerase chain reactions

limits on amplification

At the start of the polymerase Chain Reaction, the number of DNA molecules made increases exponentially

after about 20 cycles, it slowed down the increase becomes linear and plateaus

Why plateaus

• Reagent concentrations become limiting

• the enzyme denatured after repeated heating

• DNA in high concentrations causes the single stranded molecules to base pair with each other, rather than with the primers

Gel electrophoresis

• DNA is extracted from biological material and cut into thousands of fragments of varying lengths, using restriction endonucleases

• DNA fragments are separated by length with gel electrophoresis, on an agarose gel.

• The DNA is separated into single strands by flooding the gel with alkali which is then neutralized

• the electrophoresis trough is covered with the Nylon membrane which touches the gel and picks up the DNA fragments- southern blotting

• probe is used

• a film that is sensitive to X-rays or wavelengths emitted by the luminescent probe is placed over the southern blot overnight

• a film is exposed and the auto radiograph reveals a banding pattern in which dark bands show position if the probe and the repeated sequence. Pattern is genetic fingerprint

Probe

A short piece of DNA that is labeled with a fluorescent or radioactive marker

used to detect the presence of a specific base sequence in another piece of DNA

by complementary base pairing

Restriction endonucleuses

Bacterial and a nucleus that cuts the sugar phosphate backbone of DNA molecule at a specific nucleotide sequence

Dna fingerprint

compares DNA for fingerprints

• paternity- bands in a child’s DNA fingerprints are compared with the mothers. any band shared are inherited from here, remaining bands should be the fathers

• twins: identical twins have identical bonding patterns in the DNA

• siblings- people who have been adopted may wish to confirm alleged biological siblings

• immigration

• forensic use

• phylogenetic studies

pros of DNA profiling

• does it require invasive method to obtain sample

• technique can be used on samples that are too small for blood testing

• DNA profiling can rule out non-matches in DNA samples, falsely accused

• efforts of being made to store genetic material, Before isolated groups are into mixed and lost

gel electrophoresis

more or less repeats

• more repeats, the band travels are shorter distance across the gel

• less repeats, band travels longer distance across the gel

cons of dna profiling

violation

hacking

• some people consider that any request for DNA sample is a violation of individuals right to privacy and civil liberties

• DNA profiles are held in computer databases which are vulnerable to misuse and hacking. loss of privacy.

cons of dna profiling

probabilities

access to use of data

• profiles of a probabilities, not absolutes.

• access to and use of data must carefully be regulated as with DNA sequencing. Health insurance could use it to deny coverage or claims. Information could make public.

pros of dna profiling

wrongful convictions

if:

• It is used inappropriately to influence juries or judges

• Error make occur in procedure

• People conducting tests may not be trustworthy

• DNA evidence is planted at crime scene

Recombinant DNA

DNA produced by combining DNA from two different species

transgenic

an organism that has been genetically modified by addition of a gene or genes from another species

genetic engenering

allows gene to be manipulated, altered and transferred from one organism to another, making a genetically modified organism

when genetic material of two species is combined

result is recombinant DNA

The introduced DNA in transgenic organisms is the donor DNA and the organism is the host

when a cell has incorporated a plasmid containing a foreign gene, it is a transformed

Organisms which have had genes altered or deleted are genetically modified, but are not transgenic because they don’t contain foreign genes

process of producing a protein using genetic engineering technology

• identifying location of the gene

• Isolation of DNA fragments

• Insertion of the DNA fragment into a vector/plasmid

• Identification of the host cell that’s taken up the gene, using gene markers

• Cloning the transformed host cells

Locating the gene

A donor molecule of human DNA contains the gene that codes for insulin

The gene is identified with a gene probe (specific segment of a single strand DNA that is complementary to a section of the gene)

isolating the gene

isolated by two enzymes restriction endonuclease or reverse transcriptase

using restriction endonuclease

Bacterial enzymes that cuts DNA as specific nucleotide sequence

As the sequence occurs in many places the DNA is cut into small fragments and individual genes can be isolated

bases pair with complementary sequences very readily and are sticky ends

sticky ends

A sequence of unpaired basis on a double stranded DNA molecule that readily base pairs with a complimentary stand

bacteria E. coli produces restriction endonucleases EcoR1

catalyses the formation of breaks in the DNA backbone in specific sequence of nucleotides

The line of cut is staggered and leave sticky ends when the cut strands are separated. The four unpaired basis at each end of the two strands are in reverse order

drawbacks of restriction and a nucleus to excise a gene

• If the recognition sequence occurs within the gene of interest, the gene will be broken into fragments that have no function

• using the whole gene means introns are present and will be incorporated into the plasmids. any protein translated will contain extra amino acids representing the intron sequences and won’t be functional.

reverse transcriptase

enzyme that catalyse a the synthesis of cDNA from RNA template

method doesn’t have the problem of introns because the cDNA is made from mRNA from the cytoplasm.

The RNA in the nucleus that has been transcribed from the DNA has been processed to remove introns so they don’t appear. DNA polyermase then catalyses the synthesis of cDNA, making a double stranded molecule containing the gene for insulin

vectors

a virus or plasma used as a vehicle for carrying foreign genetic material into a cell

plasmid

small circular loop of self replicating double stranded DNA in the bacteria

making a recombinant plasmid

to isolate plasmids, Same restriction enzyme is used. The bacteria containing plasmids are treated with:

• EDTA to destabilise the cell walls

• Detergent to dissolve the phospholipid cell membrane

• sodium hydroxide to make alkaline environment that denatures the membrane proteins

plasmids can be sepreated from the cell debris

Circular DNA molecule making up the plasmid is cut open using the same restriction endonuclease to isolate the gene

same nucleotide sequence in its sticky

The vector and the gene are mixed and the complementary basic sequences base pair with each other

so the gene is now loosely bound to the plasmid

DNA ligase

enzyme that joins together portions of the DNA by catalysing the formation of phosphodiester bonds between their sugar phosphate backbones

inserting the gene into the vector

• plasmid removed from bacterial cell and cut open using restriction endonucleases

• DNA fragments and open plasmids are mixed together with DNA ligase

• insulin gene is isolated using restriction endonucleuses reverse transcriptase

• DNA ligase touches human gene to plasma at sticky ends

• recombinant plasma is taken up by treated bacterium and replicates, bacterial cells multiply fermenter to form the product

to make a good vector

• be self replicating

• Be small

• Not be broken down by host cell enzymes

• Not stimulate an immune response in the recipient

• Don’t be able to be screened to to confirm that gene was actually inserted into factor

• have markers to allow host cells to have successfully taken up the vector to be identified

transfer of DNA into the hosts cell

when plasmids are mixed with bacterial cells, The few bacteria cells take up the plasmid and become transformed

this can be increased with calcium chloride.

The positive charge on calcium ions bind the negatively charged DNA backbone of the plasma and the membrane lipopolysaccharides.

the use of genetic markers

to obtain bacteria that contains plasmids with the gene in them

the plasmid must successfully incorporate the gene and the bacteria must successfully take out plasma

• vector that has not taken up the gene of interest is empty. DNA can be sequenced.

• to identify which cells have been transformed plasmids with antibiotic resistant genes are used.

• to distinguish which transformed bacterial cells have taken up empty plasmas. blue white screening is used.

Clone

A population of genetically identical cells or organisms formed from a single cell or parent

Bacterial cells with recombinant plasmids

Eg. Containing insulin Gene are cultured in large volumes in fermenters

each culture forms a clone, produces many poppies of recombinant plasmid

when each bacterial cells replicate, the plasmids also do

Pros of genetic engineering in bacteria

• Medical products: a large amounts of pure human proteins are used to medicine

• tooth decay bacteria can make galactic acid is a major contribute of the tooth decay, modified strains don't make lactic acid and reduces cavities

• preventation and treatment of disease: modified to produce vaccines and treat disease

• enhancing crop growth

• environmental use: detecting and removing environmental hazards

Cons of genetic engineering in bacteria

• Classmates are easily transferred may exchange genes with other bacteria. If they are taken into potentially pathogenic species, infections they cause will not be treatable with antibiotics

• Fragments of human DNA is used to make Gene samples

• Microorganisms with a new Gene make become a threat if released into environment

• newly introduced genes may disrupt the normal function of others

Ways to introduce a novle Gene into a plant cell

• The Gene gun fires small spheres coated with a preparation of the Gene at plant cells.some penetrate the cell wall and are taken up through the cell membrane

• electroporation: an electric field of increases permeability of cell membranes, enhancing Gene uptake

• micro injection: membrane is pierced with an ultra fine needle and gene is injected into cytoplasm or nucleus.

• Using bacterial vector

Transforming plants with our agrobacterium tumefacians

1. A plasmid extracted from A. Tumefacians

2. restriction enzyme is used to cut the plasmid and remove tumor forming gene

3. a section of DNA containing a gene for disease resistance is located and isolated, using the same restriction endonuclease

4. a gene is inserted into the plasmid, replacing the tumor forming Gene. DNA ligase is used to join the donor and vector DNA together

5. bacterial cell is introduced into a plant cell. bacterial cell divides and gene is inserted into plant chromosome

6. transgenic plant cells are grown in tissue culture and transform plants are regenerated

Argument in favor of genetic modified crops

• Higher crop yield: introducing genes that can be resistance to herbicide is likely to decrease plant loss

• pesticide reduction: genes for pathogen resistance have additional advantage of reducing quantities of pesticide applied

• improved food

Arguments against the use of genetic modified

• Gene transfer

• pest resistance

• marked genes: genetically modified organisms contain marked genes which might concern antibiotic resistance

• biodiversity decreases

• organic farming: pollen from genetically modified crops could compromise organic crops

• economic concerns: genetically modified organisms are subject to intellectual property law and fear that associated expense

Information of genetic screening

• Confirm a diagnosis

• indicate appropriate treatment

• allow families to avoid having children with disease

• identify people act high risk of conditions that may be preventable

Concerns about genetic screening

• Belief of invasion of privacy

• defective alleles identified in prenatal tests may increase number of abortions

• individuals with defects may be placed in a high risk group for insurance purposes to cover the cost of treatment, so insurance cover is expensive and impossible to obtain

Main uses of genetic testing

Informed of results and implications

• carrier screening to identify if unaffected person carries a recessive value associated with genetic disease

• pre implantation genetic diagnosis to screen embryo generated from in vitro fertilization

• newborn baby screening

• prenatal diagnostic testing

• confirmation of suspected disease

• forensic and identity

Commercialized gene tests

Commercially available gene tests targeted at healthy people

limitations to use:

• commercial products aren't regulated or independently verified

• test only small number

• difficult to interpretate a positive result

• laboratory errors

• no available medical options for treating diseases

• provoke anxiety

• risks for discrimination and social stigmisation

Gene therapy

Technique in which defective allele is replaced with one cloned from healthy individual

provides treatment or cure

To introduce the DNA into target cells, gene therapy uses

• Virus has a vector

• plasmid as a vector

• injection of naked plasmid DNA

Somatic cell

Therapy targets body cells in affected tissues

method may be therapeutic but genetic changes are not inherited in daughter cells of treated cells

don't appear in the future generations

Germ line therapy

Introduces corrective genes into germline cells

genetic correction will be in the Offspring and in the offsprings

germline gene therapy is controversial

Duchenne muscular dystrophy

Recessive sex linked form of muscular dystrophy

Effectiveness of gene therapy

• Only a small portion of the introduced genes expressed

• they may be an immune response in the patient

Genomics and health care

Concerned with analyzing the structure and functioning of genomes

DNA is annotated, using base sequences , predictions are made about whether sequences code for RNA, proteins or have regulatory function

used to infer what metabolic pathways are controlled and genomes can be compared

Genomics and Human Genome Project

• More accurate diagnosis

• better prediction of the effect of drugs,

• new and improved treatments for diseases

• NGS technology sequences genomes very quickly and it may allow patients to have individual therapies based on their DNA sequence. Tailored therapies

Tissue engineering

Uses methods of biochemistry, cell biology, engineering and material science, to repair improve and replace biological functions

goal to produce bio artificial organs and to regenerate injured tissue in the body

Tissue engineering allows

The replacement of many tissues and organs eg. Trachea, bone, bladder and skin

Cells for tissue engineering of classified by their source

• Autologous cells are from on the same individual. The fewest problems with rejection and pathogen transmission

• Allogenic cells, come from a donor of the same species

• xenogeneic cells, from another species

• syngeneic/ isogeneic cells, from genetically identical organisms

Scaffolds

Cells are ‘seeded onto a scaffold’, it must:

• allow cells to attach and move

• deliver and retain cells and biological molecules

• be porous to allow diffusion of nutrients and waste products

• be biodegradable and be absorbed by the surrounding tissues. the rate it degrades should match the rate of tissue formation, so it will break down, leaving neotissue

Tissue culture

Cells that are grown in tissue culture form cell lines that are clones

as all the cells derived from a single parent cell are genetically identical