Module 8 - Gene technologies Flashcards
What is recombinant DNA
Contains sections of DNA from 2 types of organisms
Define recombinant DNA technology
Recombinant DNA technology involves the transfer of fragments of DNA from one organism, or species, to another
In-vivo amplification
Explain isolation of desired gene
- mRNA converted to cDNA using reverse transcriptase
- cDNA single stranded is converted to a double strand with DNA polymerase
- DNA from desired gene is cut with the restriction enzymes to form sticky ends
- Hydrolysis reaction, restriction site is specific to enzyme, palindromic, 4-6 bases
In-vivo amplification
What is a gene machine
Artificially joining small lengths of nucleotides
In-vivo amplification
Explain insertion into vector
- Vector carries DNA from one organism to another (e.g. plasmid or bacteriophage)
- Same restriction enzyme used to cut plasmid
- Sticky ends of plasmid will be complementary to the sticky ends of the desired gene- H bonds will form (A-T, C-G)
- Join phosphodiester bonds with DNA ligase
- Recombinant DNA is formed
- Add promoter and terminator region
- Add a marker gene (e.g. fluoresence or antibiotic resistance)
In-vivo amplification
Explain transformation and identification
- Return plasmid into bacteria- bacteria will become transgenic
- E.g. calcium salts and heat shock, microinjection, electroporation, liposomes, conjugation etc.
- Identify using the marker gene e.g. UV light to identify fluoresence or add antibiotic to kill bacteria that are not resistant
2 marks
Recombinant DNA technology can involve the transfer of fragments of human DNA into bacteria. The bacteria are then used to produce human proteins.
Give two reasons why bacteria are able to use human DNA to produce human proteins.
- The genetic/DNA code is universal
- The mechanism of transcription is universal
- The mechanism of translation is universal
2 marks
Suggest and explain one reason why bacteria might not be able to produce every human protein.
- Cannot splice pre-mRNA, so cannot remove introns
- Do not have Golgi(apparatus, so cannot modify and package proteins
- Do not have transcriptional factors required, so cannot carry out transcription/produce mRNA
1 mark
Explain the purpose of the marker gene.
- Shows that the gene has been taken up by cells
- Shows transgenic/transformed cells
- Allows detection of genetically modified cells/organisms
2 marks
Describe how the DNA is broken down into smaller fragments.
- Restriction ezymes
- Cuts DNA at specific base sequence/ brakes phosphodiester bonds
2 marks
Describe the roles of two named types of enzymes used to insert DNA fragments into plasmids.
- Restriction enzyme to cut plasmid
- Ligase joins DNA to plasmid
2 marks
Scientists manufactured large quantities of human insulin using genetic engineering.
They started by isolating mRNA from pancreas cells. From this they produced DNA which coded for insulin.
Suggest two reasons why it was better to start with mRNA from pancreas cells rather than with the DNA from these cells.
- Amount of mRNA > amount of DNA / multiple copies of mRNA
- Insulin mRNA is found in pancreas cells
- Introns removed in mRNA
2 marks
Describe the role of restriction endonucleases in the formation of plasmids that contain donor DNA.
Cut donor DNA, to remove gene / length of DNA
Cut donor DNA and plasmid with the same enzyme that cuts at the same base sequence
Sticky ends single strand
Attachment complementary strand
1mark
Describe the role of DNA ligase in the production of plasmids containing donor DNA.
Join/ anneal phosphodiester bonds
2 marks
Some human DNA was cut into separate pieces using a restriction enzyme which produced a staggered cut. A scientist wanted to insert these pieces of DNA into plasmids and used the same restriction enzyme to cut the plasmids. Explain why the pieces of human DNA would be able to join to the cut DNA of the plasmids
- Sticky ends of plasmid is complementary to sticky ends of desired gene
- H bonds form between complementary base pairing A-T, C-G
4 marks
Describe and explain how the polymerase chain reaction (PCR) is used to amplify a DNA fragment.
- Requires DNA fragment DNA polymerase, DNA nucleotides and primers
- Heat to 95 °C to break hydrogen bonds and separate strands
- Reduce temperature so primers bind to DNA/strands
- Increase temperature, DNA polymerase joins nucleotides and repeat method
2 marks
The polymerase chain reaction (PCR) can be used to obtain many copies of a particular gene.
Explain how the strands of DNA are separated during the PCR.
- By heating
- To break the H-bonds between complementary bases
1 mark
In a particular PCR, two different primers are added to the DNA.
Why are primers required?
To allow the DNA polymerase to attach
1 mark
In a particular PCR, two different primers are added to the DNA.
Suggest why two different primers are required.
Because the sequences at the ends of the target sequence are different / one is at the beginning and one at the end
2 marks
Explain one way in which the polymerase chain reaction differs from DNA replication in a cell.
- uses heat
- to separate strands
OR - PCR replicates pieces of DNA
- because DNA has been cut
OR - primer added in PCR
- to initiate replication
6 marks
The polymerase chain reaction (PCR) can be used to produce large quantities of DNA.
Describe how the PCR is carried out.
1 DNA heated to 95°C
2 strands separate
3 cooled / to temperature below 70°C
4 primers bind
5 nucleotides attach
6 by complementary base pairing
7 temperature 70 - 75°C
8 DNA polymerase joins nucleotides together
9 cycle repeated
BHA version
PCR- In-vitro amplification
- Add DNA nucleotides, tAq DNA polymerase and primers
- Primer:
1. Short section of single stranded DNA
2. Initiates replication.
3. Marks beginning and ends of the part of DNA needed for attachment of enzymes or nucleotides
4. Keep strands apart. - TAq DNA polymerase is thermostable- high temp does not break hydrogen/ ionic/ disulfide bonds
- Heat to 95C to break H bonds and seperate strands
- Cool to 65C to allow primer to bind and DNA nucleotides to attach by complementary base pairs (A-T, C-G)
- Heat to 72C to anneal phosphodiester bonds- optimum temp for DNA polymerase
- Repeat cycle many times. Total DNA- start value x 2^n (n- number of divisions)
1 mark
Explain the role of reverse transcriptase in PCR
Converts mRNA to cDNA
1 mark
Explain the role of DNA polymerase in PCR
Joins nucleotides to produce DNA
2 marks
Any DNA in the sample is hydrolysed by enzymes before the sample is added to the reaction mixture. Explain why.
- To remove any DNA present
- As this DNA would be amplified
1 mark
Suggest one reason why DNA replication stops in the polymerase chain reaction.
Limited number of primers/nucleotides
2 marks
Scientists have used the RT-PCR method to detect the presence of different RNA viruses in patients suffering from respiratory diseases. The scientists produced a variety of primers for this procedure. Explain why.
- Base sequences differ
- Different complementary primers required
1 mark
In the polymerase chain reaction, DNA is heated to 95 °C and nucleotides, enzymes and DNA primers are added to the mixture.
Explain why the DNA is heated to 95 °C.
- to separate the two strands of the DNA
- to break the hydrogen bonds
1 mark
What are DNA primers?
short section of single stranded DNA
1 mark
Why are DNA primers added during the polymerase chain reaction?
- to mark beginning and ends of the part of DNA needed for attachment of enzymes or nucleotides
- Keep strands seperate
1 mark
What is the advantage of the enzyme used in the polymerase chain reaction being thermostable?
- would not be denatured
- must be heated to 95 °C / must withstand high temps
2 marks
Recombinant DNA technology can involve the transfer of fragments of human DNA into bacteria. The bacteria are then used to produce human proteins. Give two reasons why bacteria are able to use human DNA to produce human proteins.
- The genetic code is universal
- The mechanism of transcription is universal
- The mechanism of translation is universal
1 mark
Suggest and explain one reason why bacteria might not be able to produce every human protein.
Cannot splice pre-mRNA, so cannot remove introns
Do not have Golgi apparatus, so cannot process/modify proteins
Do not have transcriptional factors required, so cannot carry out transcription/produce mRNA
2 marks
Recombinant DNA technology can involve the transfer of fragments of human DNA into bacteria. The bacteria are then used to produce human proteins.
Give two reasons why bacteria are able to use human DNA to produce human
proteins.
- DNA code is universal
- The mechanism of transcription is universal
- The mechanism of translation is universal
1 mark
Suggest and explain one reason why bacteria might not be able to produce every human protein.
bacteria- prokaryotes, human- eukaryotes
Cannot splice pre-mRNA, so cannot remove introns (no intron splicing in prokaryotes)
OR
Do not have Golgi apparatus, so cannot process/modify proteins
OR
Do not have transcriptional factors required, so cannot carry out transcription/produce mRNA
