5: DNA Technology

Question 1

Describe genetic fingerprinting

Answer 1

DNA obtained
PCR makes many copies of areas that contain repeated sequences, primers are used that bind to either side of these repeats so the whole repeat is amplified
Restriction endonucleases used to cut DNA
Electrophoresis separates strands according to size
DNA fragments are transferred from the gel to nylon membrane
Radioactive DNA probes are added
Developed using X-ray film (autoradiography)

Question 2

How do you improve accuracy of genetic fingerprints used in paternity tests?

Answer 2

Compare are higher number of places on the genome

Question 3

Uses of genetic fingerprinting x3

Answer 3

Determining genetic relationship
Determining genetic variability within a population
Forensic science

Question 4

When is genetic fingerprinting useful in genetic diagnosis?

Answer 4

When the specific mutation isn’t know or where several mutations could have caused the disorder, because it identifies a broader, altered genetic pattern

Question 5

Why are two people unlikely to have the same genetic fingerprint

Answer 5

The probability of two individuals having the same number of sequence repeats at each place they’re found is very low

Question 6

Role of short tandem repeats in genetic fingerprinting

Answer 6

Some junk DNA consists of repetitive, non coding base sequences
The number of times they repeat differs in each individual
The number of times the sequence is repeated at different places in their genome can be compared
Probability of two individuals having the same number of sequence at each place is very low

Question 7

Role of reverse transcriptase

Why is it useful?

Answer 7

Makes DNA from RNA
Useful as many cells only contain two copies of each gene, making it difficult to obtain the target gene. But they can contain many mRNA molecules

Question 8

What is meant by a palindromic sequence and why are they useful?

Answer 8

Sections of DNA that consist of antiparallel base pairs (base pairs that read the same in the opposite direction)
Restriction endonucleases recognise these sequences and cut the DNA at these places.
Sometimes the cut leaves sticky ends and sometimes blunt ends

Question 9

Name the components of the PCR reaction mixture and explain their function

Answer 9

The DNA sample - may be a section cut by restriction endonucleases
Free nucleotides - which will form the copied DNA
Primers - short pieces of DNA that are complementary to the bases at the start of the fragment you want, allow DNA polymerase to copy DNA
DNA polymerase - enzyme that binds nucleotides with phosophodiester bonds

Question 10

Describe the 5 steps of PCR

Answer 10

1. Heat to 95 to break the H bonds between the two strands of DNA
2. Cool 55 to allow primers to anneal to DNA
3. Heated to 72 as this is DNA polymerases optimum temperature
4. DNA polymerase lines up free nucleotides alongside each template strand. Specific base paring means new complementary strands are formed
5. The cycle is repeated
6. The amount of DNA doubles each cycle

Question 11

Name 3 stages of in Vivo cloning

Answer 11

1. Gene inserted into a vector
2. Vector transfers the gene into host cells
3. Transformed host cells are identified

Question 12

Describe how a gene is inserted into a vector

Answer 12

The vector DNA is cut open using the same restriction endonuclease that was used to isolate the DNA fragment containing the target gene. The sticky ends of the vector are complementary to the sticky ends of the gene.
The vector DNA and DNA fragment are mixed together with DNA ligase. This joins the sticky ends together.
The new combination of bases in the DNA is called recombinant DNA

Question 13

Describe how the vector transfers the gene into host cells

a) using a plasmid vector
b) using a bacteriophage vector

Answer 13

a) Host bacterial cells are placed into ice-cold calcium chloride to make their cell walls more permeable. The plasmids are added and the mixture is heat shocked, which encourages the cells to take in the plasmids.
b) The bacteriophage will infect the host bacterium by injecting DNA into it. The phage DNA then integrates into the bacterial DNA

Question 14

During step 2 of in vivo cloning, not all the bacterial cells will posses the DNA fragments. Suggest two reasons for this.

Answer 14

• only a few bacterial cells take up the plasmids when the two are mixed together (1%)
• some plasmids will have closed up again without incorporating the DNA fragment

Question 15

Describe how transformed cells are identified

Answer 15

Marker genes can be inserted into vectors at the same time as the gene to be cloned.
Marker genes can code for fluorescence, antibiotic resistance or an enzyme

Question 16

Advantages of in vivo cloning, compared to in vitro x6

Answer 16

• Useful when we wish to introduce a gene into another organism e.g. gene therapy
• It involves almost no risk on contamination, sticky ends mean contaminant DNA will not be taken up, in vitro requires a very pure sample
• It is very accurate, as mutations are rare
• It makes copies of very specific genes
• It produces transformed bacteria that can be used to produce large quantities of gene products
• can produce modified DNA

Question 17

Advantages of in vitro cloning, compared to in vitro x2

Answer 17

• It is extremely rapid, take hours rather than days

- It does not require living cells, no complex culturing techniques

Question 18

What is a DNA probe?

Answer 18

short strands of ssDNA, specific base sequence that’s complementary to the gene you’re looking for. The probe also has a label attached, so that it can be detected

Question 19

How are DNA probes used to find locate a gene?

Answer 19

Sample of DNA is digested into fragments using restriction endonucleases and separated using electrophoresis
The fragments are then transferred to a nylon membrane and incubated with the fluorescently labelled DNA probe. If the gene is present, the DNA probe with hybridise to it. the membrane is then exposed to UV light and if the gene is present there will be a fluorescent band.

Question 20

How can the base sequence of a gene be determined by restriction mapping?

Answer 20

Different restriction enzymes are used to cut labelled DNA into fragments
The DNA fragments are separated by electrophoresis
The size of the fragments produced is used to determine the relative locations of cut sites
a restriction map of the orgincal DNA is made - a diagram of the piece of DNA showing the different cut sites, and so where the recognition sites of the restriction enzymes are found

Question 21

State the components of the 4 test tubes in the Sanger method and explain their function

Answer 21

many ssDNA fragments of the DNA to be sequenced
a mixture of nucleotides, ATCG,
a small quantity of one of the four terminator nucleotides
a labelled primer to start the process of DNA synthesis
DNA polymerase to catalyse DNA synthesis

Question 22

Describe the Sanger method and explain how it works

Answer 22

Binding of nuecleotides is random, so the addition of a normal or terminator nucleotide is equally likely
DNA synthesis may be terminated at any point so the DNA fragments in each test tube will be of varying lengths
All will end in the same base and can be identified because the primer is attached at the other end.
The fragments in each test tube are separated using gel electrophoresis and visualised under UV light
The complementary base sequence can be read from the gel.
The smallest fragment is at the bottom, each band after this represents one more base added.
So by reading from the bottom to the top, you can build up the DNA sequence one base at a time

Question 23

In the sanger method which way to you read the complementary DNA base sequence

Answer 23

From the bottom to the top

Question 24

Difference between germ line and somatic cell therapy

Answer 24

Germ line: replacing or supplementing the defective gene in the fertilised egg. this ensures that all the cells of an organism will develop normally
Somatic cell: Targets just the affected tissues, the additional gene is therefore not resent in sex cells, and so is not passed on to future generations. Treatment needs to be repeated periodically as cells are continually dying and being replaced. Long term aim is to target stem cells that give rise to mature tissues, effective in long term

Question 25

What are the two ways in which gene therapy may be used to treat cystic fibrosis?

Answer 25

gene replacement: in which the defective gene is replaced with a healthy gene
Gene supplementation: in which one or more copies of the healthy gene are added alongside the defective gene. As the added teens have dominant allies, the effects of the recessive allies of the defective gene are masked.

Question 26

Facts about cystic fibrosis

Answer 26

Caused by mutant recessive allele, example of a deletion mutation
Mutation results in one less amino acid in the CFTR protein
Cl- ions can no longer pass through protein, ions remain in cells, water remains in cells, mucus is thick and sticky
Symptoms include: mucus congestion in the lungs, less efficient gas exchange, mucus in pancreatic ducts and sperm ducts

Question 27

What does CFTR stand for?

Answer 27

cystic fibrosis transmembrane conductance regulator

Question 28

Describe the use of adenoviruses to deliver cloned CFTR genes

Answer 28

adenoviruses are made harmless by interfering with a gene involved in their replication
Grown in epithelial cells, along with plasmids that ave the normal CFTR gene
CFTR gene becomes incorporated into the DNA
These viruses are isolated and purified
Introduced into the nostrils of patients
Adenoviruses inject their DNA into the epithelial cells of the lungs

Question 29

Describe the use of lipid molecules to deliver cloned CFTR genes

Answer 29

CFTR genes isolated and inserted into plasmids
Plasmids vectors reintroduced to bacterial host cells and gene markers are used to detect transformed bacteria
These bacteria are cloned to produce multiple copies of the plasmids
Plasmids extracted and wrapped in lipid molecules to form a liposome
Liposomes are sprayed into nostrils
Liposomes pass across the phospholipid portion of the cell surface membrane

Question 30

These forms of delivery (liposomes and adenoviruses) aren’t always effective because:
x4

Answer 30

adenoviruses may cause infections
patients may develop immunity to adenoviruses
the liposome aerosol may not be fine enough to pass through bronchioles
even where the CFTR gene is successfully delivered to the epithelial cells, very few are actually expressed

Question 31

What does SCID stand for?

Describe it?

Answer 31

severe combined immunodeficiency
sufferers do not show cell-mediated immune repose nor are they able to produce antibodies
Indiviudals inherit a defect in the gene that codes for the enzyme ADA. this enzyme usually destroys toxins that kill white blood cells.

Question 32

Describe gene therapy to treat SCID

Answer 32

The normal ADA gene is isolated from human tissue
Inserted into a retrovirus
The retroviruses are grown with host cells to increase the number of copies of the ADA gene
The retroviruses are mixed with the patients T cells
The retroviruses inject a copy of the normal ADA gene, into the T cells
The T cells are reintroduced into the patients blood to prove the genetic code needed to make ADA

Question 33

Why is gene therapy to treat SCID limited?

Answer 33

T cells live for only 6-12 months so treatment must be repeated
If bone marrow stem cells are targeted this would reduce this problem but does increase the risk of leukaemia

Question 34

Effectiveness of gene therapy

Answer 34

the effect is short-lived
it can induce an immune response
the genes are not always expressed
it is not effective in treating conditions that arise in more than one gene