Revision study notes

gene technology

June 2024
1. Enzymes ..tools in genetic engineering
A
A) Three ways of obtaining gene used in genetic engineering

1. Restriction enzyme

1.Extracted from bacteria

2.Cut at a specific base sequence ( 4 to 6 base pairs ) known recognition /
restriction site
3.Cut a DNA double strand .
4.Leaving blunt ends or sticky ends …with palindromic sequence .

Uses :
1. To extract gene/ cut plasmid to be used in genetic engineering
2. To Cut genome and form DNA fragments
In gel electrophoresis
Microarrays ..comparison between genome of different species
DNA sequencing ……

Compare between Crispr cas 9 and restriction. Enzyme

1. The target sites are specific sequence of four to six bases…less precise cuts ..( restriction
enzyme )
• Crispr/Cas 9 recognise longer base sequence of up to 20 base pairs recognition site which
means
• Specific recognition sites so more precise cut

2. the base sequence within Crispr can be modified to match more sites so more g RNA can be
made than restriction enzyme .

3.restriction enzyme Cut leave blunt ends or sticky ends , crispr cas 9 Cut leaving straight ends
only.

4.crispr cas 9 can recognise many sequences at a time

2. Reverse transcriptase

• Produced by retrovirus
• Use RNA as a template to produce a sscDNA.
• By adding a DNA primer
• Then mRNA template is removed by digesting it with RNA ase
• Where the DNA polymerase make another DNA strands .
• Single stranded sticky ends are added to allow insertion of c DNA into the vector
( plasmid )
With advantage of :
1. Much easier to extract the specific mRNA and mRNA is present in many copies
2. Host cell used may be bacteria which might not have the metabolic capability of
processing RNA transcripts that contain introns ( non coding portions-of DNA) .

3. Synthesizing DNA / gene artificially from nucleotides :

Analyse the gene product ( amino acid sequence ) and then

synthesis the gene …genetic code is universal …..usually vaccines
are done in this way .
 Other enzymes used in gene technology
B

3. Ligase
Used to link together the sugar phosphate backbone of the DNA molecules
with plasmid producing recombinant DNA
Forms a phosphodiester bond between Okazaki fragments during DNA
replication…..in natural process.

4. Taq DNA polymerase

Has high optimum temperature so doesnt need to be replaced during pCR

Extracted produced from bacterium of hot spring THERMUS aquaticus .
Naturally use of DNA polymerase
For copying DNA by adding nucleotides sequentially to the DNA
template strand and repair mis matching nucleotides . synthesises,
polynu- cleotides / strands,of DNA in (semi-con- servative) replication from,
dNTPs / activated (DNA) nucleotides

2. Steps of genetic engineering ( GM bacteria / GMO )

1. Identify and isolate the gene and cut from the DNA using a specific restriction
enzyme
2. Sometimes form copies of the desired gene using PCR
3.Cut open the plasmid using the same restriction enzyme .
4. Then join them ( mix the gene with the cut plasmid ) after adding a promoter
Where they are joined by ligase to link the sugar phosphate back bone and plasmid
Producing recombinant DNA / plasmid .
5. Introduce the plasmid ( recombinant DNA ) back into bacteria ( to be
transformed ) by heat shock / plasmid is incorporated into the genome
of ..organism …
6 Gene cloning using DNA polymerase and binarry fission ( replication of GMO)
7. The gene is expressed / transcribed and translated into the protein
 3. Steps to produce a genetically modified crop which is insect resistant

1. Isolate the gene for Bt toxin from the Bacillus thuringiensis

2. Using restriction enzyme
3. Form copies of the desired gene using PCR.
4. Vector plasmid ( Ti plasmid from Agrobacterium tumefaciens) is removed from
a bacteria.
5. Cut open the plasmid using same restriction enzyme .
6. Which is mixed with the gene after adding a promoter , they are joined using
ligase enzyme ( to link the sugar phosphate back bone ) .
7. Forming recombinant DNA/ plasmid .
8. Introduce the plasmid into maize embryos to incorporate the gene into the
genome of maize
9. The gene is transcribed into protein ( Bt toxin )

D
4. Plasmids suitable vector in gene cloning

1. Small piece of double stranded DNA ( small circle of DNA ) so can be inserted into cell so
don;t contribute to much additional DNA to the host cell ..

2. They replicate in the host cell and independently from the host cell …cloning occurs ….high
copy numbers . …High copy number = get many copies of cloned genes .

3. Easy to extract from bacteria and easy to be taken by bacterial cell.

4. Have a number of recognition sites ( multiple cloning site ) so cut be cut by using different
restriction/ endonuclease enzyme .
5. DNA / gene can be inserted
6. They posses gene markers such antibiotic resistance that allow them to be readily
recognised ( help in identifying transformed bacteria i.e the bacteria that took the
recombinant DNA) .
7. Circular thus reducing host cell degradation / stable
8. Act as vector and may carry promoter so gene can be expressed / transcribed .
5. Whats recombinant DNA

Recombinant DNA
DNA that contains genetic material from 2 different organisms.

6. Promoter added to the desired gene when producing modified cell

1. Promoter is needed for switching on the gene / needed for gene

expression/ needed to initiate gene transcription .

2. Allowing RNA polymerase and TF to bind to the promoter sequence

to initiate transcription .
A) .where it allow the RNA polymerase to recognise which of the two
strands will be the template strand to be transcribed
B) determine the level of gene expressions in the right time and sufficient amount
and in the right cell .

3. Other wise
A) the gene has to be inserted near an existing promoter which hard to be done as
this might disrupt the expression of other genes
B) in case of eukaryotes , it might be inserted in any chromosome and gene may be
included within introns
C) gene might share a promoter with host gene that doesn’t get switched on in this
host cell

7. GENE markers are used to identify the host cell that have successfully taken the gene .

A) gene which causes the bacteria to be resistant to an antibiotic

Used in case of production of insulin
Tetracycline
resistance 1. Culture ( agar plate containing ampicillin antibiotic ) , where
Ampicillin
resistance
the plasmid containing resistant gene to ampicillin will cause the
bacteria with these plasmids to survive ……exclude the bacteria
that took nothing C
*

B
2. To identify the bacteria with recombinant DNA , where gene
-
coding for resistance to tetracycline has been deactivated by

t insertion of gene of interest ( gene for insulin) …then any

bacteria which had the recombinant DNA would not be able to
grow on agar plate containing tetracycline . B
B) GFP
1. Gene coding for GFP ( which is dervied from jelly fish ) which act as a gene
marker
2. Its inserted in the plasmid
Down streaming from the promoter so that both gene marker and desired gene
are transcribed
3. Apply U. V light ( ultra violet )
4. Visible green fluorescence is observed
5. With no harmful effect on host cell
+ easier to identify
1. Gel electrophoresis, microarrays , genetic engineering
C) blue white screen ..gene producing / coding for a protein / enzyme
whose action can be easily identified

Plasmid contain a gene coding for enzyme beta glucurinidase ( GUS)

( which orginates from E. Coli ) which produce a colorless substance , that is
easily stained .
8. Examples of products of genetic engineering ( proteins)

A) insulin

How can we produce insulin from genetically modified bacteria

1. Extract mRNA for insulin from pancreatic beta cells .

2. Then mRNA is incubated with the enzyme reverse transcriptase ( obtained from
retroviruses )
3. Using mRNA as a template to make cDNA
4. Then cDNA is converted into double stranded DNA molecule using DNA
polymerase to assemble nucleotides
5. Where the sticky ends are created using restriction endonuclease and the
plasmids are obtained and cut using same restriction enzyme ( EcoR1) …leaving
complementary sticky ends .
6. Then mix the insulin gene with plamsid
7. Some of the plasmid sticky ends pair up with the sticky ends of the insulin gene …
using ligase which links together the sugar phosphate backbone of DNA molecule
and the plasmid forming RECOMBINANT DNA

Advantages for producing insulin in this way

1. More effective beacuse its identical to human insulin

2. More rapid response.
3. No iummune response
4. Highly pure so no risk of infection being transferred with insulin

5. Cheaper for larger volumes

6. Has fewer ethical and moral objections because animals are
not involved in its production
7. Good for people who had developed tolerance to animal insulin
B) factor VIII ..for blood clotting

Factor VIII is called anti hemophiliac factor .

These people with hemophilia ( poor blood clotting due lack of this factor VIII) ….they need
regular injection of VIII

In old time they uses donated blood….carry risks for infections

GMO producing factor 8

Advantages :
1.Large unlimited supply :
2. No risk of infection
3. Less chance of rejection / immune response / side effects .
4. Cheap and quick to produce
5. No need to wait for donors ( over come any ethical objection that people may
have to extract and transfer blood / human material from one person to another .)

Over coming all problems that can come from blood donations
A) where extracting the factor from blood is difficult as needs many donations
to get just small amount of factor VIII
B) overcoming the risk that factor VIII could be contaminated with disease
from donor .

C) ADA enzyme

Gene coding for this enzyme ( adenosine deaminase ) which is found in

lymphocytes
Mutation in this gene …..deficiency in enzyme …SCID ( sever combined
immuno deficiency disease)
Solution use of the ADA enzyme ..to treat SCID made by genetically modified larva
of caterpillar. ….
The enzyme given to patients waiting for gene therapy or if its n’t possible to carry
gene therapy

H
9. General technique for making copies of DNA

1. its used in electrophoresis ( DNA profiling ) .

2. Genetic engineering ( genetically engineered product using a GM bacteria )
3.microarrays ..to increase amount of mRNA .
4. Same steps was used in DNA sequencing …( to produce probes …use in
bioinformatics )

1. Steps of PCR

Importance :
1. Produce large number of copies of a lengthy DNA ( amplification OF DNA )
as may be we are starting with small quantity of DNA
2. Rapid and efficient than normal DNA polymerase

Tools :
1. Thermal cycler
2. Buffer
3. Taq DNA polymerase produced by Thermus aquaticus .
4. Primers
5. dNTPs ( deoxy ribonucleotide triphosphate )
Primer:

1.The primer anneals to single stranded DNA ( denatured )

2.Using complementary base pairing
3.Formation of hydrogen bonds ( between primer and DNA strand )
4.provides the staring sequence for Taq polymerase to bind and begin DNA
copying .
5.Because the Taq polymerase can attach only to double stranded DNA .
6. Prevent rejoining of the two separated DNA strands .

Steps
1. large number of copies of a length of DNA / amplification of DNA.
2. rapid and efficient than normal DNA polymerase.
3. A small sample of DNA needed.
4. Taq polymerase (obtained from bacterium living in hot springs Thermus aquaticus
so the enzyme is very tolerant to heat i.e thermostable).
5. the temperature is increased to 95C causing the 2 strands of the DNA fragments
to separate as hydrogen bonds are broken...DNA denatured..s
6. The primers are added.
7. The mixture is cooled to 65C causing the primers to join ( anneal) to their
complementary bases at the end of the DNA fragments .
8. The primer provides the staring sequence for Taq polymerase to begin DNA
copying .
9. The temperature is increased to 72 C. Optimum temperature for the Taq
polymerase
( has high optimum temperature) to add complementary nucleotides along each of the
separated DNA strands. Building up a new complementary DNA strands .
10. Once the two DNA strands are completed , the process is repeated by carrying
out the temperature cycle again ... Taq polymerase has high optimum temperature so
doesn’t need to be replaced each cycle .......allowing DNA amplification.

Its an auto mated process

 10. Gel electrophoresis Used in analysis of proteins and DNA

For DNA
Separating of DNA by length( DNA profiling / genetic finger printing )

A) for proteins
Use polyacrylamide
Steps :
Poly acrylamide type of gel used in case of electrophoresis of protein

1. Poly acrylamide is the type of gel used

2. The charge on protein molecules depends on the ionisation of the R groups
of amino acids ( NH3+ and coo- _
Depending on pH
So during application keep the pH constant using a buffer solution .
3. Steps :
-before applying to the gel to lose their tertiary and form a linear structure …
they can be separated on basis of overall charge .
• use buffer solution to maintain pH .
-Apply on gel ( poly acrylamide type pf gel in case of electrophoresis of
proteins )
-Apply electric current field ,,,where the gel electrophoresis separate the
protein fragments
-Proteins move towards the anode
…. being more positive so move more slowly
…. move for shorter distance
-. Looking at position of bands
-Looking at number of bands / size of bands
-. Compare the peptide bands with standard / refernce bands
-state conclusion
 B) of DNA ( genetic fingerprinting / DNA profiling )

VNTRs Separates fragments into length order

Each individual has a unique genome and therefore a unique DNA profile or genetic
finger print Relies on short , repeating sequences of DNA found in non
coding region called introns
Cointain repetitive sequence of DNA called VNTRs( variable
number tandem repeats )
With every individual , the number and length of VNTRs has a unique pattern

Which are different in different individuals except identical twins

Steps of DNA electrophoresis

1. Extraction from saliva, drop of blood , hair

2. Amplified using PCR to be visible ion agrose gel

3. The DNA is cut into fragments by restriction enzyme , cut at recognition sites
producing fragments of different length . ( leave VNTRs intact)
4. Gel electrophoresis
A. The DNA fragments are loaded into wells in agrose gel at the cathode end
B. Add buffer solution to maintain pH so as not to affect net charge
C. Apply electric current which causes an electriec field that cause the separation of
DNA fragments .
D. With the negatively charged DNA move towards the anode
E. The smaller the fragments , the faster they move so over all separation of
fragments according to their length order .

5. DNA fragments are separated to produce single strands using southern blotting .
6. Use DNA probes
Which fluorescence in U. V light
So banding pattern can be visualized and compared with reference DNA in terms of
position and size

Explain how the presence of gene for ……can ne be confirmed once

electropherisosis is complete
 Explain how the presence of gene for ……can be confirmed once
electropherisosis is complete .

Electrophoresis ….compare bands …share a recent ancestor

After electrophoresis Same answer

DNA probe used ,

Which fluorescence in U.V
So can be visualized
So banding pattern can be visualised and compared with reference DNA
in term of position and size

11. DNA sequencing

PCR+ electrophoresis

DNA sequencing is used to determine the precise sequence of base pairs

in a length of DNA

Steps
1. Cut into fragments ..restriction
2. Denature to separate the two strands
3. The primer is added
4. Cooling at 55C , anneal the primer
5. 72 C for taq DNA polymerase
6. Producing copies of different lengths of gene
7. Gel electrophoresis separate the fragments
8. Allow detection of gene fluorescence
9. Sequencing of bases read by computer and then compared .
To show closely related species using DNA sequencing

1. Sample
2. Fragments
3. heat ( denaturation )
4. Primer
5. Cooling
6. Heat for taq DNA polymerase
7. Producing COPIES of different lengths

Gel electrophoresis
1. Detection of bands / gene fluorescence
2. Base sequence of genes
3. Compare bioinformatics ( software with large data base amino acid
sequence / base sequence )
4. Compare similarities and differences …more accurate .

Electrophoresis :
1. Compare recent ancestor
2. Genetic Finger printing / DNA profiling ….closely related or not
Compare the bands size and position
3. Can be used to confirm the presence of a specific gene …
Fluorescent probe , UV light
Radioactive probe , X ray
12. Microarray

A technique used …….in two ways :

1. Comparing the gene present in complete genomes of two different species
( gene analysis )
2. Finding out which genes are expressed in tissues or cells at any given
time ( gene expression profiling)

Gene expression profiling ….tool to determine whether the DNA for a

particular individual contains mutation in genes

Microarrays …gene analysis ( not DNA profiling )

Analysis the genome and see which genes are expressed in each species

1. Compare
1. Short lengths of ssDNA probes are attached to microarray
2. The probes are complementary to the alleles / DNA being tested ( each probe is unique to a
particular gene / allele )
3. Were with each individual spot ..has many copies of the same DNA probe

4. DNA / alleles collected from each species , cut into fragments and denature to give lengths
of single stranded DNA ( ssDNA) .
5. The target DNA is labelled with fluorescent tags …….each species has a specific color ..red ,
green

6. The labelled DNA samples are mixed together and allowed to combine ( hybridise ) with DNA
probes on microarrays

7. Any DNA that doesn;t bind to probes on microarrays is washed off .

8. The microarray is then inspected using ULTRA VIOLET light ( causing hybridised probes /
tagged to fluoresce.)
2. Finding out which genes are expressed in tissues or cells at any given time
( gene expression profiling)

1. The mRNA from the two types of cells is extracted and collected .
2. And then use reverse transcriptase using mRNA as a template to produce cDNA
3. In case the amount of mRNA in a cell at one time is quite small , the quantity of cDNA
can be amplified by PCR.
4. The cDNA is labelled with fluorescent tags ….denatured to give single stranded DNA
and allowed to hybridise with probe ion microarrays .
5. Each probe is unique to a different gene .
6. Spots on the microarray that fluoresce indicate the genes that were transcribed in the
cell ( indicating genes expressed )
7. The intensity of light emitted by each spot indicate the level of activity of each gene ….high
intensity indicates that many mRNA were present in the sample…..and vice versa ( light
intensity is proportional to degree of expression) …..thus knowing activity of gene which is
needed in treating cancer .
 1. Describe how microarrays /bioinformatics can be used to
invetsigate the genetic basis of ….disease
2. Mutation can result in blindness..how can u identify the gene with
mutation . To help
3. Microarrays can be used to predict risk of diseases …identify the couples
genes with mutation take a
gene analysis
decision
1. Use microarray technique to be able to identify the active / switched on
genes / transcribed genes
Then add steps of microarray ( gene expression profiling )

2. Then collection and analysis using bioinformatics …analysis of genome

of cells with and without disease

3. Compare the sequence of the active genes to identify the mutation

13. Bioinformatics

Bioinformatics : collection, processing , analysis of biological

information and data using computer soft wares ( data bases) , Fast ,
accurate

Advantages
1. Large data base
2. Fast , accurate , efficient
3. Use data base to find probes / to compare /match other sequences
4.Using computer software
5. Statistical analysis
6. Facilitate access to large amount of data
7. Facilitate access to data on DNA and protein.
8. Store / anaylse / process info
9. To calculate / measure quantity of similarity and differences

12;46
 Whole gene sequencing Microarray

A process of determination of A process of detection of gene

Definition
the complete DNA sequence expression of pre defined genes
of living organism genome using DNA probes and microscopic
slide

Sequencing base technique Principle

Hybridization based technique

Identifies all sequences in a genome Results Identifies the presence of pre defines
sequence

More expensive compared to

Cheaper than whole genome
micro array Price
sequencing

Generate a huge amount of data so needs

Defines presence or absence of pre defined
a relevant storage capacity and ( known ) sequence so no need for a significant
computing power huge storage capacity or computing power
Good idea question related to DNA sequencing ?
14. Crispr Cas 9
General ways for treatment/ cure :
A) Crispr cas 9 …cure
B) genetic engineering …to produce protein …treatment ..temporary
C) gene therapy ….cure / but sometime if not done on stem cells it has to be
repeated
D) RNAi ..knocking down gene …not cure

Definition :
A form of genetic engineering in which the genome of an organism can be changed by
deleting , inserting or replacing a length of DNA using a method such as the crispr/
Cas9 system .

Crispr cas 9 as idea ( read to understand )

Crispr …..base sequence in bacteria codes for gRNA

g RNA direct endonuclease Cas 9 enzyme towards specific base sequence
gRNA bind to complementary base sequence on length of DNA

Cas9 cut DNA by breaking sugar phosphate back bone of DNA

bacteriophage ( virus )
Knowing that cas 9 has 2 active sites to cut DNA across both strands .

Cas protein can integrate part of viral DNA by inserting them into crispr
sequence ..retaining memory

Disadvantage
Ethically have some concerns including its permanent
modification in genome …that keeps inherited .
15. Mechanism of Crispr cas 9

1. In lab scientists creat a strand of guide RNA that matches the base sequence need to
be edited .
2. gRNA find the faulty allele …as gRNA has a complementary base sequence to the
faulty allele
3.Cas9 is added to the gRNA , where Cas 9 forms a complex with gRNA
4. Direct Cas 9 to the faulty allele
5.Cut the sugar phosphate back bone of BOTH DNA strands and remove section of
DNA
6. The DNA repair mechanism can be done
7.
A) in case of treatment of dominant genetic disorders
-adding one or more nucleotides ( a base sequence is inserted ) to silence the allele/
deletion or removal or inactivation of mutant allele …so stop production of mal
functioning protein
A) a stop codon could be introduced into the base sequence ;(inactivation)
so mRNA is produced but no functioning protein is translated ;
B) if there is an insertion of a base or a deletion of a base, a frameshift occurs ;(deletion)
all codons downstream of break in DNA are changed ;
so that a protein with a very different amino acid sequence is produced that is non-
functional ;

B) in case of recessive genetic disorders :

-inserting a short length of prepared double stranded DNA with specific correct base
sequence inserted into the broken DNA for repair mechanism / to correct faulty allele
-or inserting a complete functioning gene .
By forming phosphodiester bond
So DNA / gene restore the correct sequence of bases
Coding for a functional protein
 Problem A Problem B

• Dominant allele • Recessive allele

• Cause disease • Causes a disease
• Code for mal functioning protein • Doesn’t code for protein / non
• Solution ….silencing functioning protein
• insert a base sequence to silence the gene • Solution ..correction
A) deletion or insertion for frame shift • A) short length of double stranded
B) stop codon DNA to correct faulty allele
• B) insert a complete functioning
gene

Treatment
16. Why we cant treat Huntington disease by gene therapy :
Gene editing
Why cant treat huntington disease by gene therapy …
Deletion to the repeats
allele is dominant ….
( CAG )
so still expressed even when normal recessive allele is present …
Where we cant replace dominant allele . …………
Dominant allele affects the tissues in different body parts
gene therapy used only to treat recessive allele disorders .

17. Why we can treat cystic fibrosis / recessive genetic disorder by gene therapy
In case of recessive genetic diseases
A) recessive allele often, codes for a non-functioning protein ;
b) any allele inserted ( dominant allele ) into a cell should produce a
functioning protein ;
In case of dominant genetic disease
dominant allele codes for a malfunctioning protein ;
dominant allele needs to be, ‘switched off ’ / ‘silenced’, which is
difficult ;
gene therapy cannot target the exact location in a chromosome
where a piece of DNA should be inserted to disrupt an allele (e.g. by
introducing a stop codon) ;
 18.New technique controlling gene expression other than Crispr cas 9 / gene
therapy ( RNAi ) :

Silencing gene ( post transcriptional gene silencing ).

Using RNA interference ( RNA i)

Process in which RNA molecules are involved in
sequencing - suppression of gene expression

RNA interference (iRNA) is a new alternative to the methods of gene therapy described
above.
Small molecules of RNA are taken up by cells and combine with the mRNA of a gene
that needs to be ‘silenced’.
Results of a clinical trial of a drug which uses this technique were published in 2019.
The drug silenced a gene that codes for an enzyme in the pathway that produces haem
in the liver.
iRNA has none of the ethical issues associated with the techniques that involve
changing genomic DNA. Which is no permanent modification of genetic material

Crispr cas 9 RNAi

Loss of function / knock out so no residual gene Slow down gene expression ( some
activity…gene silencing residual gene function= knock down )

Effect is permanent Effect is not permenant

Specific to a particular DNA sequence / gene iRNA is Not specific to a single gene

No protein synthesis Low level of protein synthesis

Works on DNA Works mRNA

Higher cost Lower cost
Low off target High off target
Disadvantages of RNA i

Effect is not permenant

Not specific to a single gene
Slow down gene expression ( some residual gene
function= knock down )

Advantages

Non of the ethical issues associated with techniques

that involve changing the genomic DNA
19 crispr Cas 9 vs restriction enzymes

Both are endonuclease

But cut at specific recognition site / specific DNA bas sequence
Both extracted from bacteria
Both can cut making blunt ends
But restriction can make both sticky ends and blunt ends

Crispr cas 9

I
Restriction
Blunt ends
Blunt or sticky ends
Needs a gRNA
Doesnt need a gRNA
Recognise longer base
Less accurate , recognotion sequence of up to 20 base
site are of 4 to 6 base pairs ..so more precise
sequence
Modify base sequence in Crispr ,
Specific for specific recognition sites to produce more g RNA can
match more sites than restriction
enzyme

These target sites, or restriction sites, are

Notice concerning restriction enzyme specific sequences
of four to six bases.
20 genetic screening advantages as per disease

Genetic screening : analysis of person’s DNA to check for a presence of a

particular allele….to be done in adults , Fetus, embryo in uterus , embryo produced
from IVF .

1, BREAST CANCER genes :

BRCA 1 , BRCA2

Screening for ADULTS enable Early treatment

Prevent early death
Prevent unnecessary prolonged suffering
Alert person to keep up with regular check up
Needed for family support by preventing early death of parents .
Early diagnosis is cheaper than treatment
Life style changes

2. Huntington’s gene / allele :

• Family planning and change life style

• Way of living should be advised , prepare for the future.
• Remove anxiety
• Can choose whether to have children

Disadvantage : lead to anxiety , no treatment , still he might not

develop the disease , financial discrimination ( life insurance
refusal ) .
C) cystic fibrosis
Mutant recessive allele
1.this may assist people with decision on whether to have a child or not to have a
child ..for adults
2. In case both parents are carriers for genetic condition ..so they can decide to
have fetal testing ….fetus
And if the fetus is homozygous recessive , they could be offered to terminate
pregnancies
3. Treatment can begin early .
4. Parents can be Prepared early for the cost of treatment
5. Remove any worry
6. This child can be informed to take decision about having children when older .

D) SCID 1,. Parents have the choice

2. If done after birth , Early diagnosis and treatment
3. Prevent child with SCID from developing several infections before
diagnosis
4. Removes worry if not present.
21 genetic screening advantages in general

1.This may assist people with decision on whether to have a child or not to have a
child ….…

2. where if the fetus is homozygous recessive , parents can be offered to

terminate pregnancies ./

3. treatment can begin early …which improves life expectancy

4. parents can prepare early for cost of treatment /

1. removes any worry if not present ( removes uncertainty for those with a
genetic disease in the family )

6. this child can make an informed decision about having children when older .

7. can avoid having offsprings with serious genetic disease.,

8. allow couples to have children who would otherwise choose not to due to risk
of genetic disease.
allows couples who are both carriers of a genetic condition to make decisions
about, starting a family / having more children / seeking IVF and embryo
biopsy ;
9. In case of Huntington’s disease ….allow Family planning and change life style
Way of living should be advised , prepare for the future.
10. Identifies carriers of genetic conditions
11 allows people to plan ; e.g. organise care / take out medical insurance / make
will / gift property to children / retire early.
 22 genetic screening disadvantages in general ( both ethical and social )

1. Risk of false positive or negative result ( inaccurate )

2. Healthy child might be aborted if they are given a false positive result .
Ethical
3. Could lead to selection based on gender or specific traits ( designer babies)
4. ethical issue(s) raised by termination of pregnancy following prenatal screening ;
. Embryos are damaged
.Embryos are the right to life

4. May conflict with religious issues.

5. Cost implication to health service or individuals ./ results may affect ability to get,
insurance / credit / jobs ;
6. Affect life insurance
7. genetic test may not be test for all mutations ;
8. results may cause, anxiety / stress / panic / depression (if positive)
9. DNA analysis not available for everyone ;

Different ideas for different questions

Social implications

Social and ethical implications of screening embryo

Same answer as above
1. Risk of false positive or negative result ( inaccurate )
2. Healthy child might be aborted if they are given a false positive result .
3. Could lead to selection based on gender or specific traits ( designer babies)
. Embryos are damaged
.Embryos are the right to life
4. May conflict with religious issues.
5. Cost implication to health service or individuals .

Pros : can avoid having offsprings with serious genetic disease., allow couples to
have children who would otherwise choose not to due to risk of genetic disease.
Discuss ethical reasons why parents using IVF may not choose to have PGD

1. Risk of false positive or negative result ( inaccurate )

2. Healthy child might be aborted if they are given a false positive result .
3. Could lead to selection based on gender or specific traits ( designer babies)
. Embryos are damaged
.Embryos are the right to life

How genetic screening could reduce the number of cases of genetic diseases?

1. Identifying the embryos with recessive alleles ( carriers)

2. If embryo has allele for genetic condition abortion can be a choice
3. They can use they IVF and select unaffected embryos to implant
4. Or the woman can choose not to have children at all

23 Methods of genetic screening

A) prenatal screening ..on fetus B) pre implantation genetic diagnosis ..on embryo
1.Amniocentesis Implications
. Chorionic villus sampling
For -Allow couples to have children ,
Advantages :
who would else decide not to have
1. Reduce feeling of worry ( - ve
kids .
result )
-Avoid having offsprings with
2. Early treatment ( +ve result )
genetic disorders .
3. Decide to terminate
Against Avoid late abortions
pregnancy
Discard ( remove ) embryos with genetic diseases
Disadvantages Which is ethically unaccepted as embryos are
Spontaneous abortion potential life
Done later in deciding termination Besides this might be a conflict with some religious
would be traumatic as it will be taken beliefs
at late time so terminat
Gives a chance to Selection of gender and specific
traits
24 Steps of genetic screening on fetus

1. Samples taken from fetal DNA ( extracyed from maternal blood )

2. Samples of fetal DNA sequenced
3. Analysis of the fetal genome
A) Number of chromosomes
Ex Down syndrome with extra chromosome number 21

B) Structure of chromosome
Deletions / translocations
Example : Emanuel syndrome
Extra piece of chromosome 11 attached to chromosome 22

C) Single gene disorder

Example cystic fibrosis mutation in allele for CFTR protein ( CFTR gene)

25 Examples of genetic disorders as cystic fibrosis

Normal

1. Normal CFTR gene

2. So CFTR can function normally
3. Cl- ions move out through CFTR protein and into the mucus
4. Sodium channels inhibited by CFTR protein so Na+ ions remain outside in mucus
5. Water move out of epithelial cells into mucus by osmosis.
Resulting in thinner mucus so cilia can beat and sweep mucus away from air ways

Disease

1. Mutation in gene coding for CFTR channel protein

2. So CFTR cant function properly
3. Cl- ions build inside cells
4. water cant move out cell by osmosis …mucus become thicker
5. Cilia can’t beat to sweep thick sticky mucus
6. Bacteria has optimum condition-to grow and replicate ..cause infection
7. Block sperm duct and pancreatic duct
26 Gene therapy steps

1. Obtain the functional allele

2. Insert the allele in a virus vector
3. Remove the target / stem cell
4. Insert the allele / virus into target / stem cell
5. Return the target cells into the body

Gene therapy in case of introducing gene coding for ADA

Obtain normal allele for ADA enzyme

Insert into a virus vector ( adeno associated virus )
Extract the stem cells / target cells which are bone marrow cells .( use
drugs before extraction to increase number of stem cells in bone
marrow )
Insert the allele for ADA into the target / stem cells
Stem cells are injected into the blood

27 Limitation of gene therapy

1. Gene therapy can be temporary if not done on stem cells so needs to be repeated
2. If the allele is inserted into the genome of the cell , some time it might not be
expressed Or deactivated Or it might affect the expression of other genes.
3. Only recessive conditions such as hemophilia , and LCA ( leber congenital
amaurosis ) can be treated
4. Use of a virus as a gene vector Small packaging capacity /small amount of DNA
can be carried
5. Lower ability of virus to integrate the gene into the host’s genome
6. Can cause cancer / allergic response / infection / immune response.
7. Virus may not enter the target cell
28 Examples of diseases treated by gene therapy

1. LCA : Leber congenital amaurosis

Recessive mutant allele cause cells in the retina to die off from early age

Recessive allele cause the disease lead to impaired vision from birth leading to complete
blindness in early adult hood

They reverse the action using gene therapy for people who are homozygous recessive

The dominate allele RPE65 , code for a protein ………..regenerate the visual
pigments in rods and cone cells in retina after they have been exposed to light .

Eye good organ for developing gene therapies because :

1. Its small and easy to target
2. There is a little activity of the immune cells inside the eye, so low risk of harmful
immune response to vector .

1. ADA SCID Isolated in plastic bubbles to be protected from infections

Adenosine deaminase
Severe combined immunodeficiency

First : T lymphocytes removed from diseases child

Normal allele of ADA gene was introduced inside these lymphocytes
using a virus as a vector

Yet it needed to be repeated every 3 to 5 months

 Second : Stem cells from bone marrow
Introduce the healthy allele using retrovirus
Limitation ….the children experienced leukemia
Reason ….as result of the retrovirus used as a vector
Which inserted their genes into host’s genome randomly ….means they might
insert their genes within another gene or into a regulatory sequence which might
activate a gene causing cancer

Solution was using AAV ( adeno associated virus ) which doesnt

insert into genome in the host’s cell genome

29 Advantages of using editing over gene therapy

Advantages of editing over therapy

Gene editing is more precise
No risk of immune response , cancer , allergies
No need to add a promoter
Gene editing is a permanent solution , that involves the editing of the patients
own gene rather than inserting / introducing a gene

30. Compare between gene therapy and crips cas 9

Gene therapy Crispr Cas 9

Virus vector X

X gRNA+ Cas 9
( endonuclease )
Recessive disorders Both recessive or dominant disorders

Temporary and has to be repeated Permanent

Gene might not be taken by host cell / Gene can be deleted / silence …silence
might be inserted somewhere that affects transcription …so no mal functioning protein
the expression of another gene / not Or can insert precisely a functioning gene to be
expressed transcribed into a functioning protein
31. Social and ethical considerations of using gene therapy

1. Gene therapy is unlikely to offer treatment to all disorders such as heart

disease , high blood pressure and diabetes ( social )
2. Treat only recessive disorders ( social )
3. Cells in the respiratory tract are short lived and therefore any
successful CF therapy developed in futures would have to be taken constantly
( social)
4. Development of health problems such as inflammation
5. No right to interfere with gene ( ethical ) .

32. Role of genetic counselor

1. Couple who are carriers to the genetic disease or having a family members
of genetic disorder, older women , history of recurrent of miscarriage.
,………..so they carry genetic screening and go to the counseller

Add details of genetic screening

Prenanatal screening
using amniocentesis Or chorionic villus sampling
Pre implanatation genetic diagnosis
Or use a family pedigree .

2. Counsellers they’re trained experts that provide help / increase awarnnes of diseases and
how to get prepared and manage them in an a-family.
3. Help in taking decision according to ethical , social and religious issues
A) may discuss termination of preganancy
B) may discuss the effect of having affected child on existing siblings
C) may discuss financial implications
D) may discuss treatments
E) may discuss ethical issues .
 Genetically modified crops

Pest / insect resistance

Explanation

1. Bt toxin is toxin which is lethal to insects ( insecticidal toxin ) that eat it but hrmless to other animals .
2. Thus lead to less use of pesticides
3. Where gene coding Bt toxin irs taken from a bacterium , Bacillus thuringiensis .
4. And transferred to crop plants to make them resistant to insect pests .
Complications :
'
1. Insect population can evolve resistance to toxins .( Bt toxin can accelerate the evolution of resistance to it )
2. Damaging effect on other species of insects.
3. The transfer of the added gene to other species of plant
Steps to produce a genetically modified crop which is insect resistant

1. Isolate the gene for Bt toxin from the Bacillus thuringiensis

2. Using restriction enzyme
3. Form copies of the desired gene using PCR.
4. Vector plasmid ( Ti plasmid from Agrobacterium tumefaciens) is removed from a bacteria
5. Cut open the plasmid using same restriction enzyme .
6. Which is mixed with the gene after adding a promoter , they are joined using ligase enzyme
( to link the sugar phosphate back bone ) .
7. Forming recombinant DNA
8. Introduce the plasmid into maize embryos to incorporate the gene into the genome of maize
9. The gene is transcribed into protein ( Bt toxin )

1.Gene coding for beta carotene is obtained

2. using restriction enzyme for both cutting the gene and
the plasmid
3. Then mix the gene with plasmid to form recombunant
DNA using ligase enzyme
4. Then insertion of the gene into the rice cells
5. Where the gene is transcribed and then translated to
produce enzyme that makes carotene .
Herbicide resistance

Gene giving resistance to the effect of herbicide glyphosate '

So allowing weed control by spraying during growth of the crop

1. Where the herbicide glyphosate inhibits an enzyme involved in synthesis of three amino acids ( phenylalanine ,
tyrosine and tryptophan .

Explanation
These three amino acids are needed to produce proteins essential for growth .

Glyphosate is absorbed by a plant’s leaves and then transported to growing tips

So without production of proteins in the growth areas , the plants dies

2. The gene was taken from a strain of bacterium Agrobacterium

3. Concerns :
. GM plants are likely to become an agriculture weed when they grow into field of other crops
. Pollen will transfer the resistant gene to wild relatives of the crop plant producing hybrid offspring that are
invasive weeds
. Herbicide resistant weeds will evolve because so much of the same herbicide is used .
Reduce biodiversity
Genetically modified animals

GM salmon

GH regulatory gene from a Pacific Chinook salmon + got a promoter from another species

Explanation

Injected into a fertilised egg of Atlantic Salmon

GH produced through the year ( achieving ' full potential of the

gene) instead of just being produced in spring and summer

Modified salmon reach market size 18 months rather than 3 years for unmodified oned

Dont
G Modified salmon reduce their ability to compete with wild salmon in '
natural environment ….so FDA declare that they are less likely to have any
significant effect on the environment and they are safe as food .
Explain the significance of cereal crops in human diet

1.High carbohydrate content

2.Source of energy
3.Proteins provides amino acids For growth
4.Low in fat ( 2 to 4%)
5.Contains essential fatty acids
6.Source of vitamin B / vitamin E
7.Golden rice is rich in vitamin A as vitamin A deficiency causes blindness .
8.Contain wide range of minerals
9.Such as calcium for bone development
10. High in fibres for peristalsis

Bad ….
Might alter the taste and may be unsafe for human
 Social

Advantages of using plants that have been Disadvantages of using plants that have been genetically
genetically modified modified

1. Increase food production. 1. GM seed could be difficult for farmers in developing

2. Improve food quality countries to obtaIn.
3. Add nutrients to crop to improve human health. 2. High cost of buying GM seed...too expensive for farmers
4. Crops may be more tolerant to climate changes to buy…may have to buy seeds every season .
5. Crops can be grown in poor quality land without the 3. May not grow well in all conditions .
need of fertiliser….gm crops adapted to unfav confitions. 4. Toxicity of more herbicide left after use / might have
6. Pest/ insect/ fungal disease resistance thus allergic reactions in human….may be unsafe to human
increasing crop growth. 5. Under developed countries becoming more dependent on
7. Less pesticide used. other countries.
8. Cost effective to farmers 6. Cross pollination with wild plants
9. Less effect on food chains and pollinators ..so 7. New more resistant weeds(super weeds)
beneficial to environment. 8. Loss of traditional variety
10. Herbicide resistance reduces competition from 9. Loss of genetic diversity
weeds 10. Less food available for other animals so affecting rest
11. Example: Golden rice for extra vitamin A of food chain.
11. 11. May have to buy seeds every season
 Read only

There are several claims for the benefits of using GMOs in food production and many arguments
against their use. Some of these are:
1. GM technology gives plant and animal breeders the ability to introduce Features that could never be
introduced by selective breeding.

2. The use of GM technology speeds up the changes needed to ensure crops and live stocks can survive
and produce high yields as environments change because of climate change , so ensuring food security.

3. Farmers can’t keep for sowing for the following crop as GM crops do not ‘breed true” ; this favours large
scale commercial farmers and does not favour many farmers in developing countries.( i.e GM seed could
be difficult for farmers in developing countries to obtain)

4. Genetic engineering is an expensive technology sometimes carried out By large companies whose
main priorities may be profit rather than any environmental economic consequences.

5. We are dependent on seven types of grain producing crop plants . These are all becoming genetically
uniform . A loss of genetic diversity may mean that we will be unable to alter these plants if there are
unforeseen future environmental challenges.