4.5 - Application of reproduction and genetics

Question 1

kPurpose?

Answer 1

to improve knowledge and understanding of genetic disorders
and improve diagnosis and treatment

Question 1

Human Genome Project?
1990-2003
Information shared with private sector

Answer 1

It is sequencing the nucleotides in the human genome

Question 2

Aims?

Answer 2

Identify all the genes on the genome including the location on the chromosome

Determine the sequence of 3 billion base pairs and store on a database

Used in the private sector to develop medical innovation - whatever found - able to research

investigate ethical, legal and social issues

Improve tools for data analysis

Question 3

3 findings?

Answer 3

Only 20,500 genes on the genome - less than anticipated

more repeated segments of DNA than anticipated

less than 7% of proteins are specific to vertebrates
emphasising a close relationship between living organisms

Question 4

Sanger sequencing?

Answer 4

DNA is broken into single stranded fragments of different lengths
(800 bases)
complementary strands were synthesised but were incomplete because 4 nucleotide triphosphates were altered
(NTP)

Question 5

For each NTP?

Answer 5

For each NTP, the deoxyribose was removed to make
dideoxynucleotide
this was incorporated into the newly synthesised strand to prevent DNA polymerase binding to the next nucleotide so the chain length could not increase (terminated)
Last nucleotide was marked with a radioactive isotope (fluorescent)
from each original DNA fragment , a large number of complementary DNA strands could be produced

Question 6

How were strands separated

Answer 6

were separated by gel electrophoresis according to size

Question 7

100K genome project?
NHS
2012

Answer 7

they studied 100,000 NHS patients with cancer or a red disease

Question 8

Aims?

Answer 8

To study variation within the human genome
develop the UK genomic industry
use genomics in healthcare to benefit patients
enable medical and scientific discovery

Question 9

Moral and ethical concerns?

Answer 9

1) ownership of genetic information
(knowing you are predisposed to heart disease could affect insurance and cause stress)

2) If a DNA sequence is known to suggest a specific ancestor, it can lead to social discrimination

3) no company should make a financial profit from making DNA sequences

4) Should parents have access to their children’s DNA?

5) Genetic counselling for families with a history of genetic defects

6) Embryos from IVF can be used to study genetic conditions such as cystic fibrosis

7) Desirable characteristics
can be selected, such as sporting ability or intelligence ( Eugenics)

8) Storage + security of genomic data could be a concern due to hacking

9) discrimination and social stigma could outweigh the benefits of testing

10) Malaria would have been]

Question 10

Genetic fingerprinting?
(DNA profile)

Answer 10

99.9% of the human genome = the same in every person
the 0.1% makes an individual unique and is known as non coding DNA
Human genome consists of exons and introns
Introns are noncoding DNA and these sections are known as short tandem repeats and make an individual unique

Question 11

PCR?

( multiply DNA from the scene of a crime )
find some DNA but not enough)

Answer 11

polymerase
chain
reaction

Question 12

PCR?

Answer 12

uses semiconservative replication in a test tube
the DNA sample is dissolved in a buffer and mixed with the enzyme DNA polymerase, nucleotides and short lengths of DNA called primers (6-25 bases)

Question 13

Method?

Answer 13

DNA polymerase is taken from the bacterium
thermus aquaticus -
lives in hot springs and hydrothermal vents
has an optimal temp of
80 degrees, where it can remain active for 9 minutes before denaturing

The original DNA is heated to 95 degrees which separates it into 2 single strands
( 2 separate strands )

the solution is cooled to 55 degrees and the primers anneal to the complementary bases on the DNA strand

The solution is heated to 70 degrees and the DNA polymerase catalyses the 2 Strands and forms a phosphodiester bond of the sugar phosphate backbone which produces 2 identical double strands of DNA

steps are repeated many times after 40 cycles, billions of copies of the original DNA can be produced

Question 14

Anneal def?

Answer 14

stick to the surface

Question 15

Where can you take samples of DNA from?
( 2 columns)
Invasive
Non invasive

Answer 15

Invasive
Blood
Organs
Teeth
Tissue
Breast milk

Non invasive
Sperm
Hair
Urine
Skin
Saliva

Question 16

Gel electrophoresis?

Answer 16

method of separating DNA fragments according to size
The gel is made from agar rose which is a polysaccharide extracted from seaweed and has pores within it DNA is loaded into a well ( hole)
at one end of the gel
a voltage is applied across the gel + the phosphates in the DNA backbone have a negative charge and are attracted to the anode
small fragments of DNA move through the pores and migrate through the gel faster
the DNA extracted from a sample has been cut using restriction endonuclease
electrophoresis troph is covered with a nylon cloth which touches the gel and picks up the DNA fragments
( southern blotting )
Radioactive DNA probes
with complementary sequences attach by base pairing
any unbound probes -
are washed off
a film sensitive to x rays is placed over the southern blot and when exposed, produces an autoradiograph

produces a banding pattern / DNA profile
that u get on a DNA profile
this is how u make it

Question 17

Limitations of PCR?
( thermocyler)

Answer 17

contamination
any DNA entering the system can be amplified

error rate
DNA polymerase can insert the wrong base into a nucleotide once in every 9 thousand nucleotides
after 30 cycles, error rate increases to 1 in 300

sensitivity?

phenolics containing plant material
blue dye in denim
Humic acid in archaeological specimens

Question 18

Limitations of amplification?

Answer 18

enzymes denature on repeated heating
if DNA is in high concentrations the base pairs are more likely to pair w each other than the primer

Question 19

Uses of DNA profiling in society?

Answer 19

Paternity
DNA from a white blood cell - used to make a DNA profile which is compared with the mother and child

Twins - monozygotic twins - have identical banding patterns

Immigration Visas are dependent on proof of relatedness

Phylogenetic studies - different taxa can be compared to determine whether they have been classified correctly and to determine their genetic relatedness

Dizygotic twins - do not and the test is performed at birth to distinguish which type of twin they are

Question 20

Pros of DNA profiling?

Answer 20

It is non invasive
as the samples are obtained from mouth swabs, urine or hair
technique can be used on samples that would be too small for blood testing
has reversed wrongful convictions
genetic material from people around the world has been collected so that isolated groups are not lost

Question 21

Cons of DNA profiling

Answer 21

some people consider a request for DNA as a violation of the individual’s right to privacy + civil liberties
Databases are vulnerable to misuse and hacking
profiles are probabilities not absolutes
( not 100% accurate )
Data must be regulated as health insurance and car insurance companies could use it to their advantage
gender reassignment can be made public without consent
Profiling may produce wrongful convictions if used inappropriately by judges and juries
DNA evidence may be planted at a crime scene

Question 22

Genetic engineering def?

Answer 22

the transfer of a gene from one organism to another
so the gene is expressed in a new host cell
which is described as transgenic
recombinant DNA
DNA produced by combining DNA from 2 different species

Question 23

GE?

Answer 23

allows genes to be manipulated, altered and transferred to create a genetically modified organism
genes can be transferred to :
1) bacteria - to make product such as insulin
2) to plants to acquire new characteristics such as herbicide resistance
3) to humans to reduce the effect of genetic diseases such as DMD

Question 24

basic steps of genetic engineering?

Answer 24

isolate the DNA fragments
insert the DNA fragments into a vector
transfer the vector into a suitable host
use a gene marker to determine if the host cell has taken up the gene
clone the transformed host cell

Question 25

Locating a gene?

Answer 25

a donor molecule of human DNA
contains the gene which codes for insulin
locating the gene in 20,500 genes = difficult + every cell only contains 2 copies
the gene can be identified using a gene probe
a specific segment of single strand DNA that is complementary to a section of the gene
this can be done in 2 ways :
with 2 different enzymes
reverse transcriptase
restriction endonuclease

Question 26

Using reverse transcripase?

Answer 26

cells produce a specific polypeptide which contains many copies of functional mRNA which can be transcribed to obtain the target gene for insulin

mRNA - isolated and complementary single strands called copy DNA are produced from an mRNA template using the enzyme reverse transcriptase

DNA polymerase is used to make a double strand of DNA which will be an exact copy of the gene required

Question 27

Where is it taking place?

Answer 27

In the cytoplasm of the beta cells of the pancreas mRNA can be transcribed from the gene coding for insulin

Question 28

Advantage of this method?

Answer 28

introns have been removed from the DNA strand and the mRNA is only coding DNA
DNA polymerase catalyses the synthesis of DNA which is complementary to the single stranded mRNA
double stranded molecule can be made which contains the gene for insulin

Question 29

Using restriction endonuclease?

RE

Answer 29

re is a bacterial enzyme which cuts DNA at specific nucleotide sequences
enzyme cuts the DNA into small fragments so individual genes can be isolated
re cuts straight across the DNA double strand, making a (BLUNT CUT)
on some strands, there is a (staggered cut) which leaves unpaired bases on both strands
known as sticky ends
sticky ends = palindromes of 4 unpaired bases

Question 30

Disadvantages of using r.e?

Answer 30

the gene of interest - insulin gene may be broken into fragments when the DNA is cut
Eukaryotic genes contain introns
which would not be removed after transcription and any proteins translated will contain extra amino acids
this proteins are non functional

Question 31

Inserting the insulin gene into a vector?

vector - something that transfers something to somewhere else

Answer 31

cells do not take up genes spontaneously so the gene must be carried to the cell by a vector
viruses can be used as vectors or bacterial plasmids
Plasmids = much smaller than bacterial chromosomes and only contain a few genes
plasmids can move in and out of cells which makes them useful for introducing genes into bacteria

Question 32

Use of genetic markers?

Answer 32

to obtain bacteria
containing a plasmid
with the recombinant gene
the plasmid must successfully incorporate the gene + successfully take up the plasmid
if a vector does not take up the gene, its called empty
to identify which cells have been transformed, plasmids with antibiotic resistant genes = used
they are resistant to 3 types of antibiotic
ampicillin
tetracycline
chloramphenicol
the cells are cultured in a growth medium
containing antibiotic
if they break down the antibiotic + grow, they contain the plasmid
antibiotic resistant gene is called the marker gene

Question 33

Blue white screening?

Answer 33

to distinguish between empty plasmids + those that have taken up the gene
bacteria cells are grown on a medium of lactose analogue x-gal
they turn white if they contain the plasmid and blue if they are empty

Question 34

Purification?

Answer 34

bacterial cells containing the plasmid are cultured in fermenters
each culture forms a clone
each bacteria cell contains 40 plasmids + when the cell replicates as does the plasmids
insulin = made in large quantities + are purified for medical use

Question 35

Pros + cons of genetic engineering?

Answer 35

Pros
production of medical products
insulin for diabetes
clotting factors for haemophilia
growth hormone for dwarfism
previous products were purified from cadavers
but could transmit disease
for example HIV and AIDS

disease caused by human growth hormone - Creutzfeldt Jakob

tooth decay
streptococcus mutans makes lactic acid + causes tooth decay but modified strains do not make lactic acid and can outcompete mutans
preventing cavities
enhanced crop growth
modified bacteria can clean up mercury pollution + detect arsenic in drinking water

Cons?
plasmid - easily transferred so there is a possibility of antibiotic resistance where infections cannot be treated
activation of protooncogenes
(cancer causing genes )
a micro organism with a new gene may become a threat if released into the environment
a newly introduced gene may disrupt the normal functioning of the other genes

Question 36

Genetically modified crops?
introducing gene into the plant cell

Answer 36

a gene gun fires small spheres of gold or tungsten coated with the gene at a plant cell
spheres penetrate the cell wall + are taken up through the membrane

electroporation
electric field increases the permeability of the cell membrane - enhancing gene uptake

microinjection
membrane = pierced with a fine needle and the gene is injected into the cytoplasm

use a bacterial vector called agrobacterium tumefaciens ( soil bacteria )
to make a transgenic plant cell

Question 37

Examples of genetically modified plants?

Answer 37

soya beans
60% of the food in the UK uses soya beans
( baby food, bread + biscuits)
soya bean plants - contains a herbicide resistant gene
tomatoes
genetically modified to improve their flavour + their transportation time

Question 38

Pros and cons of genetically modifying crops?

Answer 38

High crop yield to counteract droughts + floods
pesticide reduction to protect farmland
improved nutritional value
such as golden rice which contains vitamin A
to prevent blindness
Pharming
production of pharmaceutical molecules to make antibodies, hormones + blood products for human + vetenary vaccines

pollen from gm plants can be transferred to the wild - to create superweeds

pest resistance
plants with introduced genes may resist insect attack but lead to populations of insect resistant pests

marker genes
these genes could be transferred to the bacteria in the intestines of a consumer

Question 39

Genetic screening?

Answer 39

genetic diseases cause abnormalities in the genome
single gene conditions
cystic fibrosis
Tay Sachs
( affects babies + young children and inhibits nerve function + is fatal by the age of 3 to 5 years )
common in people of Jewish descent

chromosol
down syndrome
trisomy 21

multi factorial ( MORE THAN 1 CHROMOSOME AFFECTED)
Alzheimer’s disease
genetic diseases are responsible for :
most miscarriages in developed nations
30% of post natal infant mortality in developed countries
30% of paediatric + 10% of adult hospital admissions

Question 40

Reasons for genetic screening?

Answer 40

to confirm a diagnosis
to administer treatment
to avoid having children with devastating diseases
identify people at high risk

Question 41

Reasons for not genetic screening?

Answer 41

invasion of privacy
increase in abortion rate of female foetuses
defects may place an individual in a high risk group for insurance purposes

Question 42

Commercialised gene tests?

Answer 42

targeted at healthy people
they give a probability of developing a condition

Question 43

Limitations of commercial tests?

Answer 43

not regulated or verified
only test 10% of the 20,500 genes difficult to interpret a positive result
may never develop the disease even if carrying the mutation
lab errors occur frequently such as contamination
no treatment for the genes identified
provokes anxiety
discrimination + social stigma for the findings of the test

Question 44

Gene therapy?

Answer 44

a technique of which a defective allele is replaced with a cloned one from a healthy individual, providing treatment or cure

Question 45

introducing DNA to target cell?

Answer 45

gene therapy uses 3 methods
1)virus as a vector
2)plasmid as a vector
3)injection of naked plasmid DNA

Question 46

Somatic cell therapy?

Answer 46

targets body cells in the affected tissues (CF)
method - therapeutic
and relieves symptoms
genetic changes are not inherited in the daughter cells ( cant be passing it on)
treating body cells
inhaling genes into lungs

Question 47

Germ-Line therapy?
Germinal cells

Answer 47

Introducing corrected genes into germ line cells
the genetic correction is inherited
this therapy is controversial
because introducing genes can switch on or switch off other genes or create oncogenes ( cancer causing genes)

Question 48

DMD?

Answer 48

DUTCHENNE MUSCULAR DYSTROPHY
recessive sex linked gene which effects 1 in every 3,500 male births
only effects men
DMD is caused by 1 or more deletions in the dystrophin gene
the gene has 79 exons + deletions of any alter the dystrophin mRNA
ribosome meets a stop codon too soon
dystrophin protein is not synthesised
dystrophin = a structural protein in muscle
sufferers of DND have severe muscle loss and become wheelchair bound
life expectancy = 27

Question 49

Drug treatment?

Answer 49

drisaphersen
antisense oligon nucleotide
a 50 nucleotide sequence that is complementary to the mutated sequence
it treats DND by acting as a molecular patch + binds to the mRNA over the exon with the deletion
this portion of the RNA becomes double stranded so the ribosome cannot translate it
a shorter partially functional dystrophin can be synthesised + the treatment is known as exon skipping
delivered to the patients in a sub contanious injections
clinical trials do not give clear evidence to the best age for treatments
other treatments = stem celled and gene therapy

Question 50

Cystic Fibrosis?

Answer 50

homozygous autosomal recessive allele
carriers = identified with a blood test
normal allele = transmembrane regulator
cell membrane protein that transports chloride ions out of cells
Na+ and water leave the cell by osmosis so mucus = watery and can be easily removed
mutant transmembrane regulator cannot transport ions ( Na+) and Cl- so water does not leave the cells, so mucus remains thick and sticky

Question 51

Symptoms of CF?

Answer 51

congestion due to bronchioles + alveoli becoming clogged
causing difficulty in breathing
leads to lung infection
requires physio therapy to keep airway open

Question 52

Types of stem cells?

Answer 52

stem cells can be taken from adult tissues such as bone marrow, the brain + muscle
can only be used to replace cells that are lost through wear + tear, injury or disease but cannot form all cell types
in 2006, adult cells were genetically reprogrammed known as induced pluripotent cells which can differentiate into any type of cell

Question 53

Uses of stem cells?

Answer 53

to regenerate tissues and organs
to develop insulin producing cells
repair damaged heart muscle
for spinal cord injuries, strokes + types of arthritis
stem cells are now drug testing for cancer treatment
provide models for identifying birth defects

Question 54

Advantages of stem cells?

Answer 54

have the potential to become any time of body cell
blastocysts have 100 embryonic stem cells so are easily isolated
embryonic stem cells grow easily in temperature
stem cells could reduce the risk of organ donation
unlikely to need immunosuppressant drugs

Question 55

Disadvantages of stem cells?

Answer 55

techniques are still underdevelopment and the behaviour of cells is not always predictable
technology = expensive
there are no long term studies about using stem cells

Question 56

Ethics of stem cells?

Answer 56

HFEA
Human
Fertilisation
Embryology
Authority
responsible for granting a license for research on embryos up to 14 days after fertilisation
4 requirements:
any stem cells created must be maintained indefinitely + donated to research projects or clinical therapy
stem cells must be deposited in the UK stem cell bank to increase availability
there must be no financial reward for development
donors must give consent to embryos created with their gametes

Question 57

Main issues ?

Answer 57

spare embryos from IVF
were used but these contravenes that human life should never be created as a means to an end
an embryo has moral rights but not to the same degree as a living person
the catholic church claim that life begins at conception
so an embryo should have full human rights ( the rights increase as it develops a nervous system)
14 day old embryo has no possibility of independent existence so use = justified
cloning devalues human life so reproductive cloning in the UK