Human Genetics Gene therapy & DNA fingerprinting

Question 1

what is gene therapy

Answer 1

The treatment of genetic diseases by giving patients healthy/normal functioning copies of (mutated) defective alleles.

Question 2

what are the three ways of altering the genotype?

Answer 2

• Repair of the gene
• Replacement of a defective gene with a normal one
• Addition of a normal gene leaving the defective one in place

Question 3

what are the two types of gene therapy?

Answer 3

• somatic gene therapy
• germline gene therapy

Question 4

what are the somatic cells?

Answer 4

body cells

Question 5

how does somatic cell gene therapy work?

Answer 5

Copies of a functional gene are inserted directly into the body cells of sufferers.

If the normal gene has been successfully inserted/ taken up by the affected cells and the patient later has a child, since the gene is NOT in the germ-line, the children will NOT inherit the added functioning gene. Can be short-lived and repeated treatments required

Question 6

what is a germline cell?

Answer 6

sperm/egg/zygote

Question 7

what does totipotent mean, and give an example?

Answer 7

They can divide & specialise to become any cell within the body

example: egg/sperm/zygote

Question 8

what is the germ-line?

Answer 8

The germ-line is the genetic information which is passed onto the next generation i.e. the DNA in the egg or sperm / the gametes

Question 9

how does germline therapy work?

Answer 9

A corrected / functional, gene is inserted into an egg cell that has been fertilised in vitro (IVF).

Embryo then develops, then is re-implanted into mother’s uterus.

If gene transfer is successful, mitosis ensures that every cell in the embryo contains a copy of the corrected gene.

If there are any changes in these gametes then the offspring will inherit them

it crosses generations

Question 10

what does germ-line therapy provide?

Answer 10

a treatment for an inherited condition that lasts a lifetime.

Question 11

is germline therapy legal in humans?

Answer 11

NO

Question 12

what are the problems related to gene therapy?

Answer 12

The problems are how to get the genes to the EXACT cells that need them, and then to make sure they are expressed – transcribed/translated into a protein.

Question 13

what are vectors and what do they do?

Answer 13

Vectors are carrier molecules
Need vectors to get the normal functional gene into a target cell

Question 14

what are examples of vectors?

Answer 14

Viruses
Plasmids
liposomes

Question 15

how do you use adenovirus vectors for gene therapy?

Answer 15

A functional gene is injected into an adenovirus vector, which is used to introduce the modified DNA into a human cell. 

If the treatment is successful, the new allele will make a functional protein

Question 16

what are advantages of viral vectors?

Answer 16

• Good at targeting and entering cells
  

- can target specific cell types


• can be modified so they cant replicate and destroy cells

Question 17

what are disadvantages of viral vectors?

Answer 17

• Can only carry limited size of gene
• May lead to immune response in patients which may cause side effects or block the virus getting to patient’s cells or kill cells once infected

Question 18

what are the advantages of liposome vectors?

Answer 18

Can carry normal/functional alleles as a plasmid inside the bilayer

Lipid bilayer will fuse with cell surface membrane to deliver contents directly into the cell

Can carry large genes and don’t cause immune response in patients

Question 19

why are liposomes used as gene therapy?

Answer 19

• Liposomes are spheres made of lipid bilayer
• Can carry normal/functional alleles inside the bilayer
• Lipid bilayer will fuse with cell surface
  membrane to deliver contents directly into the
  cell

Question 20

what are vector delivery methods?

Answer 20

• in vivo
• ex vivo

Question 21

what is ex vivo delivery?

Answer 21

Body cells are removed, copies of functional genes inside vectors are inserted into them and then the transfected cells are put back into the patient’s body.

Question 22

what does ex vivo delivery work best with?

Answer 22

leukemia,
patient bone marrow cells are extracted, transfected, then replaced
less likely to trigger an immune response

Question 23

what is in vivo delivery methods?

Answer 23

If a large number of body cells cannot be removed safely, functional genes inside a vector are inserted directly into the affected tissues.

Question 24

what is in vivo delivery methods used for and how?

Answer 24

cystic fibrosis treatments,

Functional CFTR gene is inserted into a liposome and inhaled to get it to the target cells which are epithelial cells of the lung

Question 25

what are the challenges in gene therapy?

Answer 25

- Gene Delivery and activation (produce functional protein) - Reduce immune response - Prevent disruption to normal host cell gene function - Commercial viability – estimated cost of developing a new pharmaceutical drug is over $500,000 – how do companies make a profit if there is a small number of patients and only 1 treatment is required?

Question 26

what vectors were used in 1993, against cf and what problems were encountered?

Answer 26

- Used adenovirus vector - Gene didn’t enter cells - Dose increased = immune response

Question 27

what vectors were used in 1995, against cf and what problems were encountered?

Answer 27

- Used liposome vector - Low levels of gene delivery into cells and DNA didn’t integrate so gene activity decreased over time - Side effects in patients (fever/inflammation)

Question 28

what genetic disorders make the best candidates for treatment using gene therapy?

Answer 28

genetic disorders caused by mutations in single genes

Question 29

what vectors were used in 1998, against cf and what problems were encountered?

Answer 29

- Used adeno-associated virus vector - No immune response/side efects - Did not enter cells effectively or integrate into genoma as expected

Question 30

What four things must a vector be able to do in order to be successful?

Answer 30

- TARGET the right cells. - INTEGRATE the gene in the cells. - ACTIVATE the gene. - AVOID harmful side effects.

Question 31

where has gene therapy shown some level of success?

Answer 31

- immune deficiencies - hereditary blindness - hemophilia - blood disease - fat metabolism disorder - cancer - parkinson's disease

Question 32

what is electrophoresis used for?

Answer 32

This technique is used to separate DNA fragments based on their SIZE so they can then be identified and analysed.

Question 33

what is electrophoresis based on?

Answer 33

It is based on the fact that the rate at which a particular fragment of DNA moves is proportional to its mass (length). The smaller the fragments will travel further than the larger ones.

Question 34

how does gel electrophoresis work?

Answer 34

- DNA Samples are placed into the wells at the negative end of the gel - Gel is immersed in a buffer solution and an electric current is passed through the solution for approx 2 hours. DNA is negatively charged, therefore fragments diffuse to the positive electrode. - Shorter lengths of DNA move faster and further. The position of fragments is shown by a dye that stains DNA - Gel electrophoresis can use either horizontal or vertical tanks to hold the gel - After separation, the position of the DNA fragments in the gel can be shown by flooding it with a DNA-binding dye that stains & shows them up

Question 35

What is the purpose of gel electrophoresis?

Answer 35

to analyse and identify nucleic acids, and to determine the purity, integrity, and fragmentation of samples.

Question 36

What acts as a filter?

Answer 36

the gel

Question 37

How is the agarose different to jell-o?

Answer 37

argarose sets firmer at higher temperatures and is more brittle

Question 38

What is the electric current for?

Answer 38

makes the DNA move

Question 39

Why are DNA fragments drawn towards the positive pole?

Answer 39

DNA molecules have a net negative charge due to their phosphate backbone, causing them to be attracted to the positively charged electrode (anode) when an electric current is applied

Question 40

what is the purpose of the loading buffer?

Answer 40

to increase the density of a sample, allowing it to sink into the well of the gel, and to provide a visible dye that tracks the progress of the sample as it migrates through the gel during electrophoresis

Question 41

What is the name of the holes left in the gel as it cools in which the DNA is loaded?

Answer 41

wells

Question 42

What does the ‘DNA size standard’ contain? What is its purpose?

Answer 42

a mixture of DNA fragments with known, predetermined sizes, which is used as a reference point to estimate the size of unknown DNA fragments within a sample being analyzed on the same gel

Question 43

How do you know when the current is running?

Answer 43

when you see bubbles forming around the electrodes in the gel chamber

Question 44

Why do the longer DNA strands migrate the least?

Answer 44

they encounter more resistance when trying to move through the gel's pores

Question 45

Why is ethidium bromide used to stain DNA? (2 reasons)

Answer 45

it intercalates between the base pairs of DNA, causing it to fluoresce brightly under UV light, making the DNA bands visible in the gel; and secondly, because this intercalation slightly alters the DNA's mobility, allowing for size estimation of the DNA fragments based on how far they migrate through the gel.

Question 46

Why is it necessary to avoid contact with ethidium bromide?

Answer 46

it is a potent mutagen, meaning it can damage DNA and potentially cause mutations, posing a significant health risk

Question 47

what is the difference between introns and exons?

Answer 47

exons are the coding regions of a gene that are used to produce proteins, while introns are non-coding regions of DNA

Question 48

What percentage of human DNA does not code for proteins?

Answer 48

95%

Question 49

where are VNTRs located?

Answer 49

Regions of non-coding DNA between genes can consist of regions of variable number tandem repeats (VNTRs)

Question 50

what are VNTRs?

Answer 50

These are repeating sequences of DNA bases between 10-80 bases long

Question 51

different repeating sequences in each person are?

Answer 51

Different repeating sequences are found at various different positions on a DNA molecule. the number of times these sequences are repeated is different in different people

Question 52

What experimental method could we use to determine how long these repeated sequences are from different DNA sources?

Answer 52

DNA sequencing using next-generation sequencing (NGS) technology

Question 53

How could we cut out these VNTR sequences from samples of DNA?

Answer 53

restriction enzymes

Question 54

The more closely related two individuals are, the VNTR?

Answer 54

the more similar the number of variable number tandem repeat (VNTR) sequences they share

Question 55

How do we carry out a genetic fingerprinting experiment? (2)

Answer 55

- The gel is immersed in alkali in order to separate the double strands into single strands. - The pattern of fragments are transferred to a nylon (or nitrocellulose) membrane by a process called Southern blotting. - Radioactive probes are used to attach to the VNTR DNA sequences (hybridisation). The probes have base sequences which are complementary VNTR repeat sequence. Any probes not bound are washed off. Areas with the probe attached are detected by x ray film (will turn it dark)

Question 56

what is southern blotting?

Answer 56

- A thin nylon membrane is laid over the gel. - The membrane is covered with several sheets of absorbent paper, which draw up the liquid containing the DNA by capillary action. - This transfers the DNA fragments to the nylon membrane in precisely the same relative positions they occupied on the gel. - The DNA fragments are then fixed to the membrane using UV light.

Question 57

what are the uses of genetic fingerprinting?

Answer 57

- solving crimes - solving medical problems

Question 58

what are the chances of two people having exactly the same DNA profile?

Answer 58

The chances of two people having exactly the same DNA profile is 30,000 million to 1 (except for identical twins).

Question 59

how is genetic fingerprinting used to solve crimes?

Answer 59

- The pattern of the DNA profile is compared with those of the victim and the suspect. - If the profile matches the suspect it provides strong evidence that the suspect was present at the crime scene (NB: it does not prove they committed the crime). - If the profile doesn’t match the suspect then that suspect may be eliminated from the enquiry.

Question 60

what can DNA profiles be used to determine?

Answer 60

DNA profiles can be used to determine whether a particular person is the parent of a child. A child’s paternity (father) and maternity (mother) can be determined.

Question 61

what can the DNA information be used in for medical problems?

Answer 61

- Paternity suits - Inheritance cases - Immigration cases

Question 62

how does a paternity test work?

Answer 62

By comparing the DNA profile of a mother and her child it is possible to identify DNA fragments in the child which are absent from the mother and must therefore have been inherited from the biological father.

Question 63

A population whose members have very similar genetic fingerprints...

Answer 63

has little genetic diversity.

Question 64

A population whose members have a greater variety of genetic fingerprints...

Answer 64

has greater genetic diversity.

Question 65

how can Genetic Fingerprinting be used For Medical Diagnoses

Answer 65

Huntingtons disease diagnosis – due to CAG repeats at the end of a gene on chromosome 4 - Fewer than 30 repeats – unlikely to develop disease - More than 38 repeats very high likelihood of developing disease - Over 50 repeats – earlier onset of disease

Question 66

how can genetic fingerprinting be used for animal/plant testing?

Answer 66

- Determine how closely related animals in captivity are to reduce inbreeding on farms or in zoos - Establish pedigree(family tree) of an animal. (Allows animal breeders to sell animals with a good pedigree for more money)

Question 67

what does inbreeding do?

Answer 67

Inbreeding reduces the gene pool and leads to increased risk of genetic disease/poor health/productivity

Question 68

Which suspect’s DNA matches the crime scene DNA?

Answer 68

the suspect whose DNA profile matches the crime scene profile