8B Genome projects and making DNA fragments

Question 1

What is the genome?

Answer 1

The complete set of DNA and genes in an organism

Question 2

What is the proteome?

Answer 2

The full range of proteins an organism can produce

Question 3

What is recombinant DNA technology?

Answer 3

Involves transferring a fragment of DNA from one organism to another
Because the genetic code is universal and because translation and transcription mechanisms are similar the transferred DNA can be used to produce a protein in the cells of the recipient organism. The recipient and donor organism don’t have to be from the same species

Question 4

What is a transgenic organism?

Answer 4

Organisms that contain transferred DNA

Question 5

What 3 ways can DNA fragments be made?

Answer 5

Using reverse transcriptase
Using restriction endonuclease enzymes
Using a gene machine

Question 6

How can a DNA fragment be made using reverse transcriptase?

Answer 6

Reverse transcriptase enzyme makes DNA from an RNA template
The DNA produced is called complementary DNA (cDNA)
mRNA is first isolated from cells
Then mixed with free DNA nucleotides and reverse transcriptase

Question 7

How can a DNA fragment be made using restriction endonucleases?

Answer 7

Some sections of DNA have palindromic sequences of nucleotides
Restriction endonucleases recognise specific palindromic sequences (recognition sequences) and cut the DNA at these places
If recognition sequences are present either side of the DNA fragment you want, restriction endonucleases can be used to separate it from the rest of the DNA
The DNA is incubated with the specific restriction endonuclease which cuts the DNA fragment via a hydrolysis reaction
Sometimes the cut leaves sticky ends which can be used to anneal the DNA fragment to another piece of DNA that has sticky ends with complementary sequences

Question 8

How can a DNA fragment be made using a ‘gene machine’?

Answer 8

A database contains the necessary information to produce the DNA fragment
This means the DNA sequence doesn’t have to exist naturally
The required sequence is designed
The first nucleotide in the sequence is fixed to a support e.g. a bead
Nucleotides are added step by step in the correct order in a cycle of processes. This involves adding protecting groups which make sure the nucleotides are joined at the right points to prevent unwanted branching
Short sections of DNA called oligonucleotides are produced
Once complete, these are broken off from the support and the protecting groups are removed
Oligonucleotides are then joined together to make longer DNA fragments

Question 9

What is in Vivo amplification?

Answer 9

Where copies of a DNA fragment are made inside a living organism

Question 10

In Vivo amplification steps

Answer 10

The DNA fragment is inserted into a vector
The vector transfers the DNA fragment into host cells
Host cells must be identified using marker genes
To produce proteins you need a promotor and terminator region

Question 11

How is a DNA fragment inserted into a vector?

Answer 11

The same restriction endonuclease that isolated the DNA fragment cuts the vector DNA
DNA fragment and vector DNA are mixed with DNA ligase in a process called ligation
This results in recombinant DNA

Question 12

What is a vector?

Answer 12

Something that’s used to transfer DNA into a cell
They can be plasmids or bacteriophages (viruses that infect bacteria)

Question 13

How are host cells persuaded to take in a plasmid vector?

Answer 13

Host cells are placed into an ice-cold calcium chloride solution to make their cell walls more permeable
The plasmids are added and the mixture is heat shocked to around 42c for 1-2 minutes

Question 14

How does a bacteriophage vector enter a host cell?

Answer 14

It will infect the host bacterium by injecting its DNA into it
The phage DNA with the target gene in it integrates into the bacterial DNA
Host cells that take up the vectors containing the gene of interest are said to be transformed

Question 15

How are transformed host cells identified?

Answer 15

Using marker genes which are inserted into vectors at the same time as the DNA fragment
A marker gene could code for antibiotic resistance
Host cells are grown on agar plates with a specific antibiotic
They divide and replicate their DNA creating a colony of cloned cells
Only transformed cells with the marker gene will survive and grow
Marker gene could code for fluorescence
Under UV light transformed cells will fluoresce

Question 16

What is a marker gene?

Answer 16

A marker is a gene that is transferred with the desired gene to enable scientists to identify which cells have been successfully transformed

Question 17

Why do you need promotor and terminator regions to produce proteins?

Answer 17

The vector needs to contain specific promotor and terminator regions
Promotor regions are DNA sequences that tell RNA polymerase when to start producing mRNA
Terminator regions tell it when to stop
Without the right promotor region the DNA fragment won’t be transcribed by the host cell and the protein won’t be made
They can be present in vector DNA or may be added in along with the fragment

Question 18

What is In Vitro amplification?

Answer 18

Uses the polymerase chain reaction (PCR)
Copies of the DNA fragment are made outside of a living organism

Question 19

What is a primer?

Answer 19

A short sequence of single-stranded DNA that has base sequences complementary to the bases at the start of the fragment being copied

Question 20

What does the reaction mixture for PCR contain?

Answer 20

The DNA sample
Primers
Free nucleotides
DNA polymerase

Question 21

PCR steps

Answer 21

DNA mixture is heated to 95c to break they hydrogen bonds between the two strands of DNA
The mixture is then cooled to between 50 and 65c to allow primers to anneal
Heated to 72c which is an optimum temperature for DNA polymerase activity
DNA polymerase lines up free DNA nucleotides alongside each template strand and joins the nucleotides together by complementary base pairing
Two new copies of the fragment DNA are formed and one cycle of PCR is complete

Question 22

How does recombinant DNA technology benefit agriculture?

Answer 22

Crops can be transformed to give higher yields or are more nutritious
So can reduce the risk of famine and malnutrition
Pest resistance reduces costs and any environmental problems associated with the use of pesticides

Question 23

How does recombinant DNA technology benefit industry?

Answer 23

Biological catalysts can be produced from transformed organisms so they can be produced in large quantities for less money which reduces costs

Question 24

How does recombinant DNA technology benefit medicine?

Answer 24

Many drugs and vaccines are produced by transformed organisms
They can be made cheaply, quickly and in large quantities

Question 25

Concerns associated with the use of recombinant DNA technology in agriculture

Answer 25

Monoculture could make the whole crop vulnerable to the same disease because the plants are genetically identical
Environmentalists are concerned about how monocultures could reduce biodiversity
Weeds that are resistant to herbicides could occur if transformed crops interbreed with wild plants. This would have unknown consequences
Organic farmers can have their crops contaminated by wind-blown seeds from nearby genetically modified crops. This means they can’t sell their crops as organic and may lose their income

Question 26

Concerns associated with the use of recombinant DNA technology in industry

Answer 26

Anti-globalisation activists oppose globalisation which could force small companies out of business
Without proper labelling some people may think there isn’t a choice about whether to consume food from genetically engineered organisms
Some consumer markets won’t import GM food and products. This can result in economic loss to producers who have traditionally sold to those markets

Question 27

Concerns associated with the use of recombinant DNA technology in medicine

Answer 27

Companies who own genetic engineering technologies may limit the use of technologies that could be saving lives
Some people worry this technology could be used unethically e.g. to make designer babies

Question 28

Humanitarian benefits of recombinant DNA technology

Answer 28

Agricultural crops could help reduce the risk of famine and malnutrition e.g. drought resistant crops for drought prone areas
Transformed crops could be used to produce useful pharmaceutical products which could make drugs more widely available e.g. in areas where refrigeration isn’t available
Medicines could be produced at more affordable rates
Has the potential to be used in gene therapy to treat human diseases

Question 29

How does gene therapy work?

Answer 29

Involves replacing or repairing defective genes with healthy ones inside cells to treat genetic disorders and cancer
If the disorder is caused by two faulty recessive alleles a working dominant allele can be added. You ‘supplement’ the faulty ones
If it’s caused by a mutated dominant allele you can ‘silence’ the allele by placing a DNA fragment in the middle of the allele so it doesn’t work anymore
Both of these processes involve inserting a DNA fragment into the person’s original DNA
The allele is inserted into cells using vectors

Question 30

What are the two types of gene therapy?

Answer 30

Somatic therapy which involves altering the alleles in body cells. It doesn’t affect the individual’s sex cells so any offspring could still inherit the disease

Germ line therapy which involves altering the alleles in sex cells. This means every cells of any offspring will be affected by the gene therapy and won’t suffer from the disease. Germ line therapy is currently illegal

Question 31

What is a DNA probe and what are they used for?

Answer 31

Used to locate specific alleles of genes
They’re short strands of DNA which have a specific base sequence that’s complementary to the base sequence of a part of a target allele
This means the DNA probe will hybridise (bind) to the target allele if it’s present in a sample of DNA

Question 32

How do DNA probes work?

Answer 32

A sample of DNA is digested into fragments using restriction enzymes and separated using electrophoresis
The separated DNA fragments are then transferred to a nylon membrane and incubated with the DNA probe
They have a label attached so they can be detected - either a radioactive label detected using X-ray film or a fluorescent label detected using UV

Question 33

What is a DNA microarray?

Answer 33

A glass slide with microscopic spots of different DNA probes attached to it in rows

Question 34

How does a DNA microarray work?

Answer 34

A sample of fluorescently labelled human DNA is washed over the array
If the human DNA contains any sequences that are complementary to the probes it will stick to the array
The array is washed to remove and DNA that didn’t stick
The array is visualised under UV light
Any spot that fluoresces means the person’s DNA contains that specific allele

Question 35

How are DNA probes produced?

Answer 35

Allele is sequenced
Then use PCR to produce multiple copies of the part of the allele - these are the probes

Question 36

What can screening using DNA probes be used for?

Answer 36

To help identify inherited conditions
Help determine how a patient will respond to specific drugs
Help identify health risks
Personalised medicine
Genetic counselling

Question 37

What is genetic counselling?

Answer 37

Advising patients and the relatives about the risk of genetic disorders
Advising people about screening and the results of their screening
Screening can help identify is someone is a carrier of a mutated allele, the type they’re carrying and the most effective treatment and prevention methods

Question 38

What is a non-coding variable number tandem repeat?

Answer 38

Not all of an organisms genome code for a protein
Some consist of VNTRs
The number of times these sequences are repeated differs from person to person so the length of these sequences in nucleotides differs too
This can be compared between individuals and this is genetic fingerprinting

Question 39

How is electrophoresis used to separate DNA fragments to make genetic fingerprints?

Answer 39

A sample of DNA is obtained
PCR is used to make man copies of the areas of DNA that contain the VNTRs
Primers bind to either side of these repeats so the whole repeat is amplified
The result is DNA fragments where the length in nucleotides corresponds to the number of repeats that a person has at a specific position
A fluorescent tag is added to all DNA fragments
Electrophoresis - DNA mixture is placed into a well in a slab of gel covered in a buffer solution that conducts electricity
An electrical current is passed though the gel. DNA fragments are negatively charged to they move towards the positive electrode at the far end of the gel
Smaller DNA fragments move faster and travel further, separating the DNA fragments according to size
The DNA fragments are viewed as bands under UV light

Question 40

Uses of genetic fingerprinting

Answer 40

Determining genetic relationships
Determining genetic variability within a population
Forensic science
Medical diagnosis
Animal and plant breeding to prevent inbreeding which would reduce the gene pool

Question 41

Down sides of genetic screening

Answer 41

May result in discrimination by insurance companies and employers