Gene Technology

Question 1

Process for recombinant DNA

Answer 1

1)identification of the desired
2)isolation of desired gene
—> using reverse transcriptase to convert mRNA to CDNA
—> cDNA + DNA polymerase makes desired gene (doubled stranded DNA )

3) restriction endonucleuases cut the DNA to create the sticky’s ends for a complementary base pairing

4) desired gene is multiplied by using PCR (DNA polymerase + DNA primers

5) plasmid is cut using the same restriction endoclease to have the same sticky end with complementary base pair

6) using DNA ligase to join the plasmid and the desired gene together
—> catalyse the formation of phosphodiester bonds between the sugar phosphate backbone of the desired gene and that of the vector DNA

7) producing recombinant DNA

8) insert recombinant DNA to the vector

Question 2

What to do after the recombinant DNA is inserted into the vector

Answer 2

Identify using marker gene to confirm that the recombinant DNA is present
—> cloning eg in the fermenter
= genetically identical

Question 3

The process of transformed organism

Question 4

What is a gene gun

Answer 4

Shoot DNA carried on very small gold or tungsten pallets into the cell at high speed

Question 5

What is liposome wrapping

Answer 5

Liposome fuse with the target cell membrane and pass through it to deliver the DNA into the cytoplasm

Advantages non invasive

Question 6

What is micro injection

Answer 6

Injecting DNA into a cell through a micropipette

Advantages
Most successful transgenic animals

Disadvantage
Inefficiency many cells have to be injected before one accepts the DNA successfully

Question 7

How does harmless virus act as a vector

Answer 7

Harmless viruses infect cells and insert their genetic material into the host cell DNA the introducing desirable DNA

Makes it useful for inserting desirable genes into human cells where the viral DNA with the new gene combines with the host DNA

Disadvantage
Viruses must be harmless and must be effective at inserting DNA in a range of hosts.

Question 8

Knockout organism : silencing gene

Answer 8

Sometimes want to remove or silence.a gene by using knockout organism
—> they do this by inserting a new gene similar to the gene to be investigated but which makes the Original DNA sequence impossible read

Furthermore, we can use knockout genes to identify the function of a particular gene

Question 9

Replica plating

Answer 9

1) the bacteria containing the desired gene also contain marker gene that make them need a specific amino acid in the nutrients medium in order to grow. Bacteria including some that have been successfully engineered to contain recombinant DNA, are grown on a master plate on a complete medium

2) the master plate is inverted and pressed on a sterile velvet surface leaving an imprint of the colonies.

3) a second plate is inverted and pressed against the velvet surface to pick up the imprint of the colonies. The medium of the replica plate lacks the specific amino acid required by the marker gene. The replica plate is incubated. The colonies that grow have not been genetically modified because they can synthesise the missing amino acid

4)the replica is compared to the master plate so genetically modified colonies can be identified and grown on

Question 10

What are marker genes

Answer 10

Genes that make a bacterium dependant on a particular nutrients or which cause the organism to fluorescent in the UV light

Question 11

Role of the knockout gene

Answer 11

1) silence gene
2) mapping identifying gene
—> don’t know the function of these genes
- knocking them out and observing the result can help to make their function clear

Question 12

Process of mass production of product

Answer 12

1)identification of the desired
2)isolation of desired gene
—> using reverse transcriptase to convert mRNA to CDNA
—> cDNA + DNA polymerase makes desired gene (doubled stranded DNA )

3) restriction endonucleuases cut the DNA to create the sticky’s ends for a complementary base pairing

4) desired gene is multiplied by using PCR (DNA polymerase + DNA primers

5) plasmid is cut using the same restriction endoclease to have the same sticky end with complementary base pair

6) using DNA ligase to join the plasmid and the desired gene together
—> catalyse the formation of phosphodiester bonds between the sugar phosphate backbone of the desired gene and that of the vector DNA

7) producing recombinant DNA

8) insert recombinant DNA to the vector

9) the modified bacterium now produced the product - the gene is expressed and cause the synthase of the product

10) the modified bacterium in placed in the fermenter and incubate it
- O2/no oxygen
-nutrients/ glucose/amino acid
-optimum temperature
-optimum pH

11) downstream processing (purifying) the product
12) pure product is produced for use

Question 13

Why is the stirrer needed

Answer 13

For evenly distributed nutrients or oxygen for the modified bacterium

Question 14

What is the sparger for

Answer 14

A device for letting air into the broth

Question 15

Transgenic plants making

Answer 15

1) Ti plasmid is extracted from A. Tumefaciens
2) gene to be carried to the plant is inserted into the Ti plasmid which is then returned to the bacterium

3) the plant is infected with modified bacterium and part of the Ti plasmid with the engineered gene become part of the plant chromosome

—> gene gun ; tiny pellets are coated with the desired DNA and then fired into the plant cell

4) A. Tumefaciens causes a tumour to develop on the plant these plant cells contain the new gene. If tumour cells are taken and cultured, whole new plants can be grown from them containing the new genes . These are genetically engineered or transgenic plants.

—> new plant containing new gene grown from gall cells

The protein can now be purified from the plant tissue or the plant can be eaten to deliver the drug

Question 16

Genetically modified animals process

Answer 16

-The gene that codes for the desired protein is injected into the nucleus of a zygote

• the zygote is implanted into the uterus of a surrogate animal where it develops into an adult animal
  —> every cell of this genetically modified animal will contain copy of the gene coding for the desired gene

The protein can be purified from eg the milk of the animal
—> human blood clotting protein can be produced from the milk of genetically modified animals

Question 17

What’s the 3 process you can use to inject the gene into the animal

Answer 17

Micro injection - where DNA is injected into a cell through a very fine micropipette

Microprojectile - DNA is shot into the cell at high speed carried on minute fold or tungsten pellets

A harmless virus can be engineered to carry a desirable gene and then used used to infect the animals cells carrying the DNA with it

Liposome wrapping - the gene to be inserted is wrapped in liposome spheres formed from a lipid bilayer these then fuse with the cell membrane and can pass through it to deliver the DNA into the cytoplasm

Question 18

Replication of RNA virus with gene technology

Answer 18

The viral RNA is used as a template by reverse transcriptase enzymes to produce a complementary strand of DNA (cDNA)

Once this single stranded DNA molecule is turned into a double stranded molecule it can be successfully inserted into the host DNA

From here it uses the host cell enzyme to produce more viral components which are assembled to form new viruses

Question 19

Define bioinformatics

Answer 19

The development of the software and computing tools needed needed to organise and analyse large amounts of raw biological data

Question 20

What is microarray

Answer 20

Used to identify active gene
—> active gene are gene that are expressed; mRNA is transcribed from them and the resulting mRNA strand is translated into a polypeptide

Question 21

Process of micro arrays

Answer 21

Microarrays have silicon or glass DNA probes attached to many spots - gene spots

—>

1) one of the mRNA is the reference source* and the other is the mRNA contains and unknown source

2) then use reverse transcriptase enzyme to convert mRNA to cDNA

3) the cDNA is labelled with flurescence labels and the reference is labelled with fluorescence labels
-reference - green
Unknown -red

4)once the reference and unknown sample is mixed together they are then allowed to hybridized with the probes on the microarrays

5)the micro arrays is then examined using ultraviolet which is scanned colours are detected by computer and information is analysed

Question 22

What does each colour such as yellow in microarrays mean

Answer 22

If the reference green and unknown red both hybridization has occurred in equal proportions then overall colour detected will be yellow

—> shows that the gene in question is being expressed in equal quantities in the reference individual and the individual from whom the unknown sample was taken

Question 23

What does each colour such as red in microarrays mean

Answer 23

If unknown samples hybrids more than the reference samples then the overall colour detected will be red

—> shows that the gene is being expressed more in the individual providing the unknown sample than in the reference individual

Question 24

What does each colour such as green in microarrays mean

Answer 24

If the reference sample hybridized more than the unknown samples then the overall colour detected will be green

Gene is being expressed more in the reference individual than in the individual providing the unknown sample

Question 25

What is microarrays used for

Answer 25

Medical diagnosis and treatment

Biotechnology

Forensics analysis

Question 26

What databases is used in bioinformatics

Answer 26

Gene sequences and amino acid
DNA, mRNA and proteins
-the database are available online and can perform analysis if the data selected

Question 27

Risk of genetic engineering

Answer 27

• concerns impact on genetically modified food organism on human health
• pest develop resistance to the modified crop defences leading to increase used of pesticides
• monoculture that are bad for biodiversity
• genetically modified crop variety are usually owned by the companies that develop them so seeds can very expensive
• some have moral objections to genetically modifying animals for the sole purpose of benefiting humans

Question 28

Benefits of genetic engineering

Answer 28

• crops can be modified to produce higher yields and have increased nutritional value reducing famine and malnutrition

-crops can be modified to be resistant to pest and reduce pesticides use; this lower production costs and decrease environmental damage

• enzyme used in industrial processes can be produced from genetically modified organism . Very cost effective
• disease can be treated with human proteins produced by genetically modified organism instead of with animal proteins this reduces the risk of allergic reaction and is more effective

-vaccines can produced in genetically modified plant tissue; these don’t need refrigeration making them more accessible to people living in rural areas

Question 29

Process transformed organism

Answer 29

1)The bacteria containing the desired gene also contain a marker gene that make them need specific amino acid in the nutrients medium in order to grow. Bacteria including some that have been successfully engineered to contain recombinant DNA are grown on a master plate on a complete medium

2) the master plate is inverted and pressed on a sterile velvet surface, leaving an imprint of the colonies. The master plate is saved

3)a second plate is inverted and pressed against the velvet surface to pick up an imprint of the colonies. The medium of the replica plate lacks the specific amino acid required by the marker gene. The replica plate is incubated. The colonies that grow have not been genetically modified because they can synthesise the missing amino acid

4) the replica plate is compared to the master plate so genetically modified colonies can be identified and grown on.

Question 30

Genetically modified animals

Answer 30

1) the gene that cods for the desired protein is injected into the nucleus of a zygote

2)the zygote is implanted into the uterus of a surrogate animal where it develops into adult animal
—>every cell of this this genetically modified animal will contain a copy of the gene coding for the desired protein

Question 31

Animal cloning

Answer 31

1)removing the nucleus from the normal body cell transferring it to another egg cell which it’s original nucleus is removed

2)a mild electric shock is used to fuse the nucleus with the new cell and trigger development

3) the newly formed cell start to develop and divide by mitosis

4) the zygote is implanted into the uterus of a surrogate animal where it develops into an adult animal