Biotechnology

Question 1

What is Morden biotechnology ?

Answer 1

Morden biotechnology refers to the processes and techniques that involved in the manipulation of DNA, cells, tissues, or biological processes to attain knowledge, produce goods and products.

Question 2

Using the production of human insulin as an example, describe the major steps in recombinant DNA technology.

Answer 2

• Obtain DNA fragments containing the DNA encoding human insulin (the gene of interest).
• Obtain vectors (e.g. plasmids).
• Cut the DNA fragments and the plasmids using the same restriction enzyme.
• Join the DNA fragments and the open plasmids together using a DNA ligase.

Question 3

What is plasmids ?

Answer 3

Plasmids are small rings of double-stranded extrachromosomal DNA

Question 4

What is the function of plasmid ?

Answer 4

As vectors for transferring gene of interests into host cells

Question 5

Name the enzyme used in the recombinant DNA technology

Answer 5

Restriction enzyme

Question 6

What is the function of restriction enzyme ?

Answer 6

~ recognises a specific base sequence and cuts the doubled-stranded DNA
~cut open the plasmids

Question 7

Why same restriction enzyme is used to cut both plasmids and DNA sample ?

Answer 7

~ restriction enzyme recognizes a specific base sequence and cuts the double-stranded DNA.
~some restriction enzyme leave short lengths of single-stranded DNA at cut ends. These cut ends are called /sticker ends
~the unpaired base in sticky ends of DNA fragments are complementary to those in the sticker ends of the plasmids

Question 8

Why same restriction enzyme is used to cut DNA samples?

Answer 8

~ cut the DNA with the same base sequence

Question 9

How do DNA fragments and plasmid blind together?

Answer 9

With the help of the DNA ligase, the DNA fragment and the plasmids joined together by hydrogen bond.

Question 10

What is the function of the DNA ligase ?

Answer 10

~ catalyses the joint of the DNA fragment and the plasmids by covalent bond (hydrogen bond)

Question 11

what is the purpose of introducing the recombinant plasmid into the hosts cells?

Answer 11

replication and expression of the DNA encoding the gene of interest

Question 12

what bacteria is usually used for the introdcing of ploasmids?

Answer 12

E.coli

Question 13

Why plasmids need to carry a selective marker ?

Answer 13

To enable selection of transformed bacteria

Question 14

how to select the traansformed plasmids?

Answer 14

~plasmids carrying a selective marker such as an antibiotic resistance gene are often used as vectors
~ when the bacteria that have been mixed with the recombinant plasmids are cultured on an agar plate containing the antibiotic, only the transformed bacteria which carrying the antibiotic resistance gene survive and divide to form colonies

Question 15

describe the process of the gene cloning

Answer 15

~ after the selection of the transformed plasmids, they are clutured in the steel tanks caklled fermenters.
~in each bacterium, ther recombinant plasmids replicates independently of the bacterial chromosome
~the plasmids is also copied into the daughter cells when bacterium divides
~ as a resuklts, many copies of the DNA encoding the gene of interest may formed

Question 16

how is the human inslulin produced using recominant plasmids?

Answer 16

~ the recombinant plasmids anre introduced to the bacteria (host cell)
~the transformed bacteria is then selected by culturing the bacteria ojn the agar plant containg the antibiotic
~the transormed bacteria are cultured in fermeters
~DNA encoding human insulin is expressed ande polypeptides are produced
~ the polypeptide is extracted from bacteria and processed into the functional human insulin

Question 17

what is the advantages using recombinant DNA technology in human insulin production

Answer 17

~insulin produced is same as the insulin produced in human body —> no rejection by the immune system
~the insulin produced is pure
~the production yield is high:
* many copies of the DNA encoding the human insulin are present in the bacterial culture.
* the growth rate is high
* the bacterial culture continuously produced insulin whereas each animal pancreas can only provide a limited amount of insulin
~ extraction cost is low

Question 18

Why do people not using pig insulin?

Answer 18

~may cause tissue rejection

~may cause disease

Question 19

How GMOs are produced ?

Answer 19

~a toxin gene is obtained and inserted into a plasmid of Agrobacterium using recombinant technology
~ the recombinant plasmid is then introduced back into Agrobaterium
~the transformed Agrobacterium is allowed to infect maize cell. During the infection, the bacterial toxin gene is integrated into DNA of the maize cell
~GM maize plants are developed from this maize cell

Question 20

What are the benefits of the production of GMOs using genetic engineering ?

Answer 20

~ “biological factories” to produce large amounts of useful products in a shorter rime and a low cost
~high productivity can increase food supplies
~have a higher nutrient value
~reduce pollution

Question 21

What is the potential hazards of the production of GMOs using genetic engineering ?

Answer 21

~long-term effects on human health is unknown
~ allergic reaction
~ superbug
~ transfer genes to wild types ( genetic pollution )
~ out-complete the wild types and reduce the biodiversity and upset the ecological balance

Question 22

What is a clone ?

Answer 22

a genetically identical copy (eg. Identical twins, plants from asexual reproduction such as vegetative propagation and cutting )

Question 23

Major step in plant clotting

Answer 23

~remove a piece of tissue from a plant and sterilise it. Put it into a sterilise culture medium containing nutrients necessary for growth
~cell divide by mitotic cell division to form a mass of undifferentiated cells called callus
~ transfer the cells of callus to culturing medium containing hormones which promote the growth of shoots and roots.
~the cells differentiate into different types of cells. Planets are formed. Grow the planets in the soil for further development

Question 24

Applications of plant clotting

Answer 24

~produce large number of plants that are economically important
~ to produce disease-free plants for agriculture
~to rescue disease-infected plants population
~to produce plants that are endangered or hard to grow from seeds or by conventional propagation
~to maintain a special breed of plants with desirable characters
~to produce genetically identical plants for research purpose

Question 25

What is the method of animal clotting in the past?

Answer 25

~ embryo splitting ~ individual cells from a single embryo are isolated and grown into embryo ~the embryo are then transferred to the surrogate mother for development

Question 26

What is the method used now ?

Answer 26

~ nuclear transfer ~a mammary gland was collected from an adult sheep ~an ovum was collected from another adult sheep and it nucleus was removed ~ the mammary gland cell was fused with ovum to form an embryo in vitro ~the embryo was implant to the uterus of a surrogate sheep to allow further development ~the surrogate sheep gave the brith to Dolly

Question 27

What is the genetically make up of dolly sheep from

Answer 27

The mammary gland cell donor

Question 28

Applications of animal clotting

Answer 28

~to propagate farm animals or endangered species ~ to mass-produce GM animals for the production of valuable pharmaceutical products or other chemicals ~to produce genetically identical animals for use in drug tests or research ~ to obtain stream cells for use in research or medicine

Question 29

What is the limitation of animal clotting

Answer 29

~ low success rate | ~ get old sooner and have shorter lifespan

Question 30

What is the advantages of cloning

Answer 30

~ desirable characters can be preserved coz’ the offspring produced have an identical genetic make-up to their parents ~ overcome reproductive difficulties of plants and animals

Question 31

Disadvantages of cloning

Answer 31

~ lack genetic variation so reduce the adaptability of the population to changes in the environment

Question 32

Limitation of recombinant DNA technology

Answer 32

Time consuming and labour intensive

Question 33

What is involved in polymerase chain reaction

Answer 33

~DNA sample, DNA polymerase, primers, nucleotides | ~ thermal cycler

Question 34

Why Taq DNA polymerase is not denatured at 90C?

Answer 34

~obtained from bacteria living in hot spring/ hot environment ~so high optimum temp.

Question 35

Process of PCR

Answer 35

~DNA denaturation: 95C high temp. So the H bond between the base pair in DNA is broken. So the double helix of DNA sample separates into 2 single DNA strands ~ Annealing of primers: 50-65C so primers can anneal to the single stranded DNA which serves as the starting point for DNA synthesis ~DNA synthesis: 70C provide the optimum temp. For the heat-stable DNA ploymerase to act. So it catalysed the addition of free nucleotides to new DNA strands

Question 36

Why primers can added to the DNA sample ?

Answer 36

~base sequence of primer is complementary to the base sequence of one end of the target region on one DNA strand ~ by complementary base pairing

Question 37

What is the cons of ploymerase Chain reaction

Answer 37

~fast for amplifying a specific region | ~ only small amount of sample

Question 38

Application of PCR

Answer 38

~ amplify DNA for diagnosis of prenatal diagnosis of genetic disease ( foetal cell from amniotic fluid) ~ amplify DNA to produce DNA fingerprinting in forensic science ( DNA sample from crime scene) ~ amplify DNA sample from the remains of historical figures or extinct species for studies ( DNA in organism degrades after they died ) ~ to diagnosis of infectious disease ( very small amount of viral and bacterial DNA can be detected)

Question 39

What is DNA fingerprinting ?

Answer 39

~ technique of using DNA analyses to identify individual

Question 40

Explain why the DNA fingerprints of these suspects showed different patterns

Answer 40

~as the no. Of VNTRs are different among individuals ~ the difference in the no of repeats result in different length of DNA fragment after cutting with suitable enzyme ~since different length of DNA fragments migrate at different speed ~different bands of DNA fingerprinting which is unique to individuals can be shown except identical twins

Question 41

What would happened if there is mutation among VNTRs?

Answer 41

~ as VNTRs are present at many different locations in the non-coding region doesn’t code for functional protein. ~ mutation in these region don’t affect the survival of organism. They can be passed on to the subsequence generations. ~ so, the no of VNTRs varies from person to person, comparing to the few variations in functional genes

Question 42

What is the method of DNA fingerprinting

Answer 42

Restriction fragment Length Polymorphism analysis

Question 43

Major step of RFLP analysis

Answer 43

~ DNA sample are cut with restriction enzyme ~ DNA fragments are separated by gel electrophoresis ~ DNA fragment are denatured into single strands and transferred to a nylon membrane ~ DNA fragments containing the VNTRs of interest are detected by radioactive DNA probes ~ The position of DNA fragments contains the VNTRs of interest are visualised by a photographic film

Question 44

Why VNTRs at 4-5 different loci on different chromosomes are usually estimated ?

Answer 44

~ to minimise the probability of 2 individuals having identical DNA fingerprintings ~ so the DNA fingerprinting is unquiet to each individual and the result is very reliable method of identifying an individual

Question 45

Limitation of RFLP analysis

Answer 45

~ time-consuming | ~ large amount DNA sample

Question 46

Why probe must be radioactive

Answer 46

~ DNA is not visible on gel ~ by binding with the probes complimentarily, the radioactivity of the probe can be easily detected shows up on the photographic film

Question 47

Applications of DNA fingerprinting

Answer 47

``` ~forensic science ~ parentage test ~ victim identification ~ authentication of Chinese medicine ~ conservation of endangered species ```