DNA fragments

Question 1

Why are DNA fragments necessary?

Answer 1

For gene technology, copies of gens are needed to work with

Question 2

Give 3 possible methods of creating DNA fragments

Answer 2

• Conversion of mRNA to cDNA using reverse transcriptase
• Using restriction endonucleases to cut fragments containing the gene from DNA
• Using a gene machine

Question 3

What is Reverse Transcriptase and its function?

Answer 3

• A group of viruses called retroviruses (e.g. HIV) contain an enzyme called reverse transcriptase
• It is used to turn viral RNA into DNA so that it can be transcribed by the host cell into proteins

Question 4

Describe the process of conversion of mRNA to cDNA using reverse transcriptase

Answer 4

• Obtain cells that produce a lot of the protein and extract mRNA that codes for the protein
• Make a complementary DNA strand (cDNA) using reverse transcriptase and DNA nucleotides
• Hydrolyse the mRNA strand
• Create ds DNA using DNA polymerase and DNA nucleotides

Question 5

Give an example of using reverse transcriptase [5]

Answer 5

• B-cells from Islets of Langerhans in the Human pancreas
• Extract mature mRNA coding for Insulin
• A single stranded complementary copy of DNA (cDNA)
  is formed using reverse transcriptase on the mRNA template
• Single stranded cDNA is used to form double stranded DNA using DNA polymerase
• This forms a double stranded copy of the Human Insulin gene

Question 6

Restriction Enzymes [6]

Answer 6

• Also called restriction endonucleases
• Bacteria contain restriction enzymes in order to protect themselves from invading viruses
• Have highly specific active sites
• Restriction enzymes are used by bacteria to cut up the viral DNA
• Usually cut DNA at specific sites - about 4-8 base pairs long, these are called recognition sites
• This property can be useful in gene technology

Question 7

Restriction Enzymes - “Blunt Ends”

Answer 7

Some restriction enzymes cut straight across both chains forming blunt ends

Question 8

Restriction Enzymes - “Sticky Ends” [3]

Answer 8

• Most restriction enzymes make a staggered cut in the two chains, forming sticky ends
• Sticky ends have a strand of single stranded DNA which are complementary to each other
• They will join with another sticky using the enzyme DNA ligase end but only if it has been cut with the same restriction enzyme

Question 9

Describe the process of using restriction endonucleases to cut fragments containing the gene from DNA

Answer 9

Restriction endonucleases cut DNA at specific palindromic sequences, either side of the desired gene

Question 10

Describe the process of using a gene machine [5]

Answer 10

• Enter the desired base sequence into a gene machine
• Check for biosafety and biosecurity
• The machine creates small, overlapping nucleotide sequences, oligonucleotides
• Join together to make the gene and replicate using PCR (polymerase chain reaction)
• Once the genes are obtained, they may then have to be modified to add promotor regions, terminator regions, or sticky ends

Question 11

In vivo cloning involved cloning vast quantities of bacteria.
These bacteria will have specific genes which have been inserted into them. What is the purpose of this?
[3]

Answer 11

• Advantageous Characteristics
• Production of Insulin
• Production of Antibodies

Question 12

How is in vivo cloning carried out? [4]

Answer 12

• Isolation
• Insertion
• Transformation
• Identification

Question 13

Isolation [3]

Answer 13

Extract the required gene using one of the following methods:
- Reverse transcriptase
- Restriction endonuclease
- Gene machine

Question 14

Insertion [6]

Answer 14

• If restriction endonucleases have not been used already, they will be used here
• This creates sticky ends, which allow the gene to be inserted
• Restriction endonuclease cuts gene from genome
• Same type of restriction endonuclease used to cut plasmid / vector
• This means both will have complementary sticky ends, as restriction endonucleases are specific to a particular DNA sequence
• Ligase joins the sticky end of the gene to those of the plasmid / vector

Question 15

Transformation [3]

Answer 15

• The modified plasmid re-enters the bacterial cell
• There is a around a 1% take up of plasmids, with even less of the bacteria proving to be useful
• A harmless, modified virus could also be used as a vector

Question 16

Identification [2]

Answer 16

• Plasmids don’t always do what you want them to, therefore, we need ways of identifying the bacteria which have successfully taken the gene/plasmid
• Marker genes are used to identify whether gene has been taken up by the plasmid, and the bacterium has taken up the plasmid

Question 17

There are several type of Marker Genes [3]

Answer 17

• Antibiotic Resistance Marker Genes
• Fluorescent Markers
• Enzyme Markers

Question 18

Antibiotic Resistance Marker Genes (replica plating) [6]

Answer 18

• A replica plating plasmid is used which has genes for resistance for 2 different antibiotics
• Plasmid cut at the site of one of these genes and the wanted gene is inserted into this space
• Bacteria is made to grow on agar with 1st (non-cut) antibiotic present
• Any bacteria from this are then transferred to different agar plates with 2nd antibiotic present
• If the plasmid was taken up with gene inserted in 2nd antibiotic bacteria will grow on the 1st plate but not the 2nd
• This will be the useful bacteria

Question 19

Replica plating is not used much nowadays due to: [3]

Answer 19

• More complicated procedure
• Antibiotic resistance genes may spread and cause resistance in other situations
• Need to look for colonies that haven’t grown on the second plate

Question 20

Fluorescent Markers [5]

Answer 20

• Plasmid created with GFP (green fluorescing protein) gene
• Desired gene is inserted into GFP gene
• All bacteria will grow, those that glow under UV light have NOT taken up plasmid
• Those that grow but don’t glow contain gene
• This is a much quicker process as it can quickly identify transformed bacteria using UV light

Question 21

Enzyme Markers [5]

Answer 21

• Plasmid created containing lactase gene
• Lactase turns a substrate from colourless to blue
• Desired gene inserted into lactase gene
• Colonies with gene inserted will not make lactase and will not turn substrate blue
• Colonies without gene inserted will turn substrate blue, and will not be used

Question 22

What is the difference between in vivo and in vitro cloning?

Answer 22

In vivo = a process that occurs within a living organism
In vitro = a process that occurs in a laboratory vessel

Question 23

Give 6 differences between in vivo and in vitro cloning [12]

Answer 23

In Vivo:
- Slower
- Uses living cells
- Allows gene expression in a different organism so a specific protein can be made
- Viral vectors have a slight risk of causing an immune response or infection
- Very accurate due to bacterial checking mechanisms
- Low chance of contamination

In Virto
- Very quick
- No cells needed
- Only DNA in sample produced
- Not inserted into living organisms so no immune system issues
- No checking mechanisms
- Contamination common, as any DNA present will be amplified

Question 24

What does PCR stand for?

Answer 24

Polymerase Chain Reaction

Question 25

What is the function of PCR?

Answer 25

To make copies of DNA fragments, increasing the size of the sample

Question 26

PCR Method [3]

Answer 26

• Reaction Mixture Ingredients: DNA sample, free nucleotides, DNA polymerase, buffer, primer
  (Taq polymerase) primers, thermocycler
• Temperature of 95°C causes hydrogen bonds within the DNA sample strands to break and strands to separate (denaturation)
• Cool to 55°C to allow primers to anneal. These allow DNA (Taq) polymerase to attach additional nucleotides (Annealing)
• Heat to 72°C. DNA (Taq) polymerase will add nucleotides to the separated DNA strands (extention)

Question 27

Why is Taq Polymerase used?

Answer 27

Taq polymerase is from an extremophile bacterium, it is used as it has a higher optimum temperature, so it does not denature at this temperature

Question 28

What is a primer?

Answer 28

Short sequence of bases complementary to the start of the sequence on the template strand, allowing Taq polymerase to bind.

Question 29

What does DNA polymerase do?

Answer 29

Short single strand of DNA with bases complementary with gene being searched for
Often have labels attached so can be identified:
- Radioactive labels
- Fluorescent labels

Question 30

What is DNA hybridisation?

Answer 30

DNA hybridisation is the process of mixing the DNA probes with a sample of DNA in order to identify if certain alleles are present

Question 31

DNA hybridisation method [6]

Answer 31

• Determine the sequence of nucleotides for the allele that is to be identified
• Create a DNA probe for this sequences, use PCR to make many copies
• Take a sample of DNA to be tested, heat to separate the strands and mix with the DNA probes
• Cool the mixture, the DNA probes will complementary base pair if the complementary sequence is present in the DNA sample
• Wash to remove any excess, unbound DNA probes
• Use X-rays to identify radioactive probes or UV light for fluorescing

Question 32

Genetic Screening [4]

Answer 32

• Genetic screening can be used to help identify whether an individual is carrying an allele for a genetic disorder.
• It is possible to identify if they are homozygous for the allele or heterozygous (homozygous will have twice as many DNA probes attached).
• This allows people to make decisions about whether to have children, or to prepare for the consequences of serious genetic illnesses.
• Oncogenes and mutations to tumour suppressor genes can be identified, so if there is a family history of cancer, anyone who has identified the alleles can be discovered.

Question 33

Genetic Counselling

Answer 33

Genetic counsellors guide people to make informed decisions about matters related to genetics that may come up in their or their families health.

Question 34

Personalised Medicine [2]

Answer 34

• Personalised medicine can be used to target different dosages or compounds as medicine depending on the genetic make-up of the person.
• e.g. Some breast cancer treatments are targeted at specific mutations so screening can be used to make sure the right medication is used.