Applications of genetics

Question 1

What method did the humane genome project use to identify nucleotides?

Answer 1

Sanger Sequencing

Question 2

Describe Sanger Sequencing?

Answer 2

• gene cut up into many different length fragments using a flourescent ‘chain terminator’ base
• If the terminator base in a ‘c’ the chain will be cut at every C in the sequence. Likewise for ATG
• The fragments are separated using gel electrophoresis and analysed for the size of fragments, revealing the base sequence of DNA

Disadvantages of Sanger sequencing:
- huge scale
- one gene at a time

Question 3

Why is Next Generation Sequencing more effective than Sanger sequencing?

Answer 3

• small scale
• can be used on smaller fragements up to 700 nucleotides
• run in parallel (many fragments sequenced at the same time)
• fast- because many reactions are done at once

Question 4

What is electrophoresis?

Answer 4

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

Question 5

What is a primer?

Answer 5

• short sequence of complementary DNA
• attaches to the start of the DNA sequence to make the area that needs to be amplified
• nucleotides bind to the primer by phosphodiester bonds

Question 6

Describe the process of PCR?

Answer 6

• DNA heated to 95 degrees to separate the two strands of the DNA molecules by breaking the hydrogen bonds between them
• temperature is cooled to 50-60 to allow primers to anneal (join) to the DNA
• The temperature is raised to 70 and a thermally stable Taq DNA Polymerase joins adjacent nucleotides on the complementary strand by phosphodiester bonds. This is termed the extention phase
• the temperature in the thermocyder is raised to 95 and the cycle is repeated. After 40 cycles there will be about a billion copies of the DNA

This is good for archeological findings to copy DNA from small samples

Question 7

How are STR’s used to create a unique genetic finger print?

Answer 7

1. DNA is extracted from the individual (PCR applied if needed)
2. DNA ‘cut’ into fragments using restriction enzymes
3. DNA is placed in wells of an electrophoresis plate. A current is applied and DNA travels through the gell to the anode (positive electrode). Shorter fragments pass through more easily and therefore travel further
4. gel is transfered to a membrane
5. the bands are made visible with radioactive probes or dye
6. x-ray shows the placement of the DNA fragments

Question 8

What does PCR stand for?

Answer 8

Polymerase chain reaction

Question 9

What is a restriction endonuclease?

Answer 9

Bacterial enzyme that cut DNA at specific nucleotide sequences. Some restriction enzymes make a blunt cut across the DNA helix, others make a staggered cut which leave unpaired bases on both strands and therefore sticky ends

Question 10

What is a sticky end?

Answer 10

A sequence of unpaired bases on a double DNA strand that readily pairs with a complementary strand

Question 11

What are the two main draw backs with using restriction endonucleases?

Answer 11

1. If recognition sequence occurs within the gene of interest it will be broken
2. eukaryotes contain introns, using the whole gene means introns will be incorporated into the plasmids. Bacteria do not contain introns so do not have the appropriate enzyme to remove them, if intron gets transcribed the protein will be non-functional

Question 12

Why does using a reverse transcriptase not have a problem with introns?

Answer 12

the cDNA is made from the mRNA from the cytoplasm, this has already been processed to remove introns

Question 13

Note: most genetic engineering notes in class book

How are plasmids isolated from bacteria?

Answer 13

• EDTA destabilises the cell walls
• Detergent to dissolve phospholipid cell membrane
• NaOH to amke an alkaline enviroment that denatures the membrane proteins

Question 14

What is used to cut a plasmid open?

Answer 14

the same restriction endonuclease as was used to isolate the gene which means it has the same nucleotide sequence in its sticky ends

Question 15

What are the four methods for introducing a new gene into a plant?

Answer 15

1. Gene gun - small particles of DNA are fired at plant cells
2. Electroporation- electric feild makes membrane more permeable to DNA
3. Microinjection- ultra fine needle is used to directly inject the gene into the plant cell cytoplasm
4. Bacterial vector- transgenic Agrobacterium tumefaciens enters plant cells and divides, introducing the gene into the cell

Question 16

Positives and negatives of genetic screening?

Answer 16

+ve- confirm diagnosis, indicate appropriate treatment, allows families to avoid having children with devastating disease, identify people at high risk for preventable conditions.

-ve - invasion of privacy, defective alleles identified in prenatal tests may increase the number of abortions, individuals with defects may be placed in a high risk group for insurance purposes to cover cost of treatment this may be expensive or unobtainable.

Question 17

What are the main uses of genetic testing?

Answer 17

• carrier screening to identify if an unaffected person carries a recessive allele assocaited with a genetic disease
• pre-implantation genetic diagnosis to screen embryos in IVF
• pre-natal diagnostic testing
• newborn baby screening
• disease conformation
• forensic and identity testing
• pre-symptomatic testing for predicting adult-onset disorders such as Alzheimers

Question 18

What are the limitations on commercialised gene tests?

Answer 18

• laboratory errors from misidentification or contamination
• commericial products are not regulated or independently verified
• difficult to interpret positive result as people who carry the mutation may never develop the disease
• they can only test a small number of the 20500 genes in the human genome
• risk of discrimination for people who have taken the test

Question 19

What is gene therapy?

Answer 19

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

Question 20

What does gene therapy use to introduce the DNA into the target cells?

Answer 20

A virus or plasmid as a vector or injection of naked plasmid DNA

Question 21

What are the two main approaches of gene therapy?

Answer 21

• Somatic cell therapy- targets body cells of affected tissues, genetic changes are not inherited by daughter cells so do not appear in future generations
• Germ-line therapy- introduces corrective genes into the oocyte, sperm of parents or into the zygote. The genetic correction will be in the offsprings gametes. This is controversial as genes interact with eachother some are switches that control other genes so influencing such genes has unpredicatable future effects

Question 22

What type of enzymes are restriction endonucleases?

Answer 22

Bacterial enzymes

Question 23

How do you ensure the host cell takes up the plasmids?

Answer 23

• addition of CaCl2 as Ca ions bind the negative DNA backbone of the plasmid to the lipopolysaccharides in the bacterial membrane
• heat shock is applied (4-43) and the plasmid DNA passes into the cells

Question 24

What is Agrobacterium tumefaciens (AT) ?

Answer 24

• A section of the bacteriums plasmid can intergrate into the plants chromosomes
• The plasmid genes are then trascribed causing tumours to grow on the plant (crown gall disease)
• The plasmid is called the Ti (tumour-inducing) plasmid. By splicing genes into this plasmid genes can be transferred to plant cells which are grown in tissue culture and regenerated into plants with express these introduced genes

Question 25

What are the negatives of gene therapy?

Answer 25

• only a small proportion of the introduced genes are expressed
• there maybe an immune response in the patient

Question 26

What is tissue engineering?

Answer 26

uses biochemistry and material science to create bio-artificial organs and to regenerate injured tissue in the body

Question 27

What are the four types of cells for tissue engineering based on their source?

Answer 27

• Autologous cells are from the same individual so least risk of rejection
• Allogenic cells come from a donor of the same species
• Xenogenic cells are from anothe species e.g from pigs, however a danger is that the pig cell contains viral sequences which are harmless to pigs but dangerous for humans
• Syngenic or isogeneic cells are from genetically identical organisms

Question 28

What is therapeutic cloning?

Answer 28

cells are grown in a tissue culture to form organs, all cells are derived from a single parent cell so are genetically identical

Question 29

What is reproductive cloning?

Answer 29

Cloning of whole organsims

Question 30

What is a stem cell?

Answer 30

An undifferentiated cell capable of dividing to give rise to daughter cells which can develop into different types of specialised cell or remain undifferentiated

Question 31

What are the four types of stem cell?

Answer 31

• Totipotent- make all cell types and cells supporting embryonic development
• Embryonic stem cells are pluripotent as they can form every cell type in the body
• adult stem cells are multipotent as they can make several cell types for example bone marrow. Replace cells lost through wear, tear and injury
• induced pluripotent- reprogrammed adult stem cells can make several cell types

Question 32

What is the potential for stem cells?

Answer 32

• tissue engineering to regenerate tissues and organs
• cell-base therapies to treat diseases - renewable resource of replacement cells e.g. for spinal chord injury
• to screen new drugs
• to develop model systems to study normal growth and identify the causes of birth defects
• investigate events in human development

Question 33

Advantages of stem cells?

Answer 33

• can become any cell type
• easy to grow in large numbers
• tissued derived from adult stem cells less likely to provoke immune response

see table on 260 for ESC V ASC

Question 34

Disadvantges of stem cells?

Answer 34

• Techniques for extracting, culturing and manipulating are still under developed
• use of stem cells is new so long term studies have not yet been possible. Concerns relating to possible unpredictable events

Question 35

What are some requirements of the Human Fertilisation and Embryology Authority?

Answer 35

Research on embryos up to 14 days after fertilisation, providing:
- any stem cells or cell lines created are maintain indefinietly to be used in many different research projects
- stem cells are deposited in UK stem cell bank so they are available to other research groups nationally and internationally
- no financial reward or development or discovery using them
- donors must give consent

Question 36

What are the issues surrounding the use of embryonic stem cells?

Answer 36

• source of the embryonic stem cells, some people aruge creating embryos for research contravenes the principle that human life should never be created as a means to an end
• Moral status of the embryo. Under Human Fertilisation and Embryology Act the embryo does have some moral rights.
• catholic church believes life begins at conception
• rights of embryo balanced against the potentially large benefits that other people may gain from the treatment/research it produces

Question 37

What are the limitations of PCR? (5)

Answer 37

see page 243