Gene Technology Flashcards
Define genome
Complete set of genes in a cell
What does recombinant DNA mean
A cell having two or more sources of DNA
What are the 5 steps in recombinant DNA technology
- Isolation of genes
- Insertion
- Transformation (transfer into microorganisms)
- Identification (gene markers)
- Growth/cloning
Why is recombinant DNA possible
Because the genetic code is:
Universal: the same triple codes for the same amino acid
Degenerate: more than one triplet for each amino acid
Non overlapping: each base is part of only one triplet
What are the three ways of isolating a copy of a specific DNA fragment
- Reverse transcriptase to convert mRNA to cDNA
- Restriction endonuclease to cut a fragment of desired gene from DNA
- Gene machine
Explain how reverse transcriptase occurs when isolating a gene
Free DNA nucleotides bind to single stranded mRNA template via complementary base pairing.
Reverse transcriptase joins DNA nucleotides together to form a single stranded cDNA molecule
DNA polymerase is required to make cDNA double stranded
Advantages of using reverse transcriptase
- Cells contain two alleles of each gene (one on each homologous chromosome)
- If the cell is expressing that gene it will contain many mRNA molecules with complementary base sequence to that gene
- MRNA is much easier to obtain
- Introns have already been removed
What are restriction endonucleases
Enzymes that hydrolyse DNA at specific base sequences. Usually at either side of a desired gene. Produce blunt or sticky ends
Gene machine
Desired nucleotide sequence is fed into a computer
Makes oligonucleotides (short sequences of nucleotides)
Oligonucleotides overlap and joined together to make double stranded DNA using PCR
Gene inserted into bacterial plasmid
Advantages of using gene machine
No introns
Artificial genes easily transcribed and translated by prokaryotes as they have no introns
What is a vector
A vector is a DNA carrier such as a bacterial plasmid or virus and is used to transfer foreign DNA into cells
Key steps of insertion of genes into a vector
- Vector DNA is cut open using the SAME restriction endonuclease that was used to isolate DNA fragment.
- This produces COMPLEMENTARY sticky ends between ends of DNA fragment and cut ends of vector DNA.
- DNA fragment and vector DNA anneal by complementary base pairings between sticky ends
- DNA ligase is used to join the DNA fragments and vector DNA at the sugar phosphate backbone.
This is called ligation and forms phosphodiester bonds
What is transformation
The process by which recombinant DNA vector is transferred into a host cell (bacteria).
Host cells which take up recombinant DNA are referred to as recombinant organisms or transformed organisms.
Why is bacteria used
Bacteria reproduce very quickly by binary fission
If foreign DNA is inserted into the organisms DNA via plasmid, it replicates the foreign DNA as well as its own.
Makes many copies of DNA fragment
Outline a method for in vivo gene cloning
- Cut gene from DNA of desired organism
- Using restriction endonuclease
OR
1.use mRNA from cell of desired organism
2. Use reverse transcriptase to form DNA
THEN
- Cut plasmid open
- Using SAME restriction endonuclease
- This produces complementary sticky ends
- DNA fragment and vector DNA anneal due to complementary base pairs
- DNA ligase joins them together
- Return the plasmid to bacteria
Why do transformed cells need to be identified
- Not all or very few vectors take up target DNA to become recombinant
- Not all or very few host cells becomes transformed
How can you identify transformed bacteria using antibiotic resistance genes
- Cells that have NOT taken up ANY plasmids will be KILLED by BOTH types of antibiotic
2 cells that have taken up the ORIGINAL plasmid will be RESISTANT to BOTH types of antibiotics - Cells that are TRANSFORMED will be RESISTANT to ONE type of antibiotic as the 2nd gene has be cut and disrupted by inserting foreign DNA.
The cells that survive the first antibiotic but destroyed by 2nd antibiotic are the transformed cells
What are the two types of way you can clone a desired gene
In vivo - via bacteria
In vitro - via PCR
What is PCR used to do
Is used to amplify (make lots of copies of DNA)
Automatically repeated many times
Number of DNA molecules doubles with every cycle making it rapid and efficient
What are the three stages of PCR
- Seperation
- Annealing
- Synthesis/elongation
Define what a DNA primer is
Short pieces of single stranded DNA with complementary base sequences to DNA fragment
What does a primer do
- Guides DNA polymerase
- Prevents DNA strands joining back together
Describe the process of PCR
- Heat DNA to 95°C which breaks the hydrogen bonds/ separate strands (denature)
- Add primers and add nucleotides
- Cool to 55 °C to allow binding or primers
- Add heat stable taq DNA polymerase
- Heat to 72 °C
- DNA polymerase joins nucleotides together
- Repeat cycles many times
Advantages of PCR
Automated - more efficient
Rapid - lost of DNA can be made
Doesn’t require living cells - quicker and less complex techniques needed
How do you stop the PCR
Primers run out
DNA nucloetides run out
Denature enzyme
How to calculate number of DNA strands after PCR
Use formula 2n (2 to the power of n)
N is the number of cycles
To calculate number of cycles from number of DNA strands:
Log2(number of DNA molecules)
What is germ line gene therapy and what are some limitations
DNA transfer to cells that produce eggs or sperm
Limitations:
- effects are unpredictable, could introduce further defects to embryo
- denial of human rights
- potential abuse - can enhance favourable characteristics and suppress unfavourable ones resulting in “designer children”
What is somatic gene therapy and what are the limitations
DNA transfer to normal body tissue
Limitations:
- not all cells take up new DNA
- not all cells express DNA allele
- only some tissue types are accessible
- body can produce an immune response to vector
Define gene therapy
A mechanism by which genetic diseases are treated or cured by changing a faulty gene with the insertion of a functioning allele.
What is a gene probe
A short, single stranded DNA molecule with a complementary base sequence to DNA fragment to be located which is radioactive or labelled by a fluorescent molecule
What do we use DNA probes
Used to locate specific alleles of genes and to screen patients for heritable conditions, drug responses or health risks
Process of locating specific alleles of genes using DNA probes
- DNA sequence must be known to then create the DNA probe
- This can be determined by using DNA sequence techniques
- Fragment of DNA can be produced using a gene machine
- Fragment can then be amplified using PCR
- Add fluorescent label which emits light under UV
- After hybridisation, DNA is washed so any unbound DNA probes are washed away
- Presence of fluorescence label indicated that allele is present in patients DNA
What is DNA hybridisation
When the DNA probe binds to target DNA because its complementary. DNA is now half fluorescent probe and half is the patients DNA
Describe how genetic fingerprinting is carried out
- DNA extracted from sample
- DNA cut using restriction endonuclease
- Must produce blunt ends and leave VNTRs intact
- Separate DNA using gel electrophoresis
- By putting DNA into a well and applying an electrical current to them
- Make fragments single stranded by adding alkaline solution
- Put nylon on top to absorb DNA
- Add probe to hybridise target DNA
- Identify using x-ray film
Describe how DNA is replicated in a cell.
• DNA strands separate / hydrogen bonds broken;
• Parent strand acts as a template / copied / semi-conservative replication;
• Nucleotides line up by complementary base pairing; (Adenine & Thymine etc)
• Role of DNA polymerase: joins adjacent nucleotides on the developing strand via condensation and formation of phosphodiester bond;
• 5’ to 3’ direction
• Each new DNA molecule has 1 template and 1 new strand
• Formed by semi-conservative replication.
Why is the DNA heat to 95°C during PCR?
• Produce single stranded DNA
• Breaks WEAK hydrogen bonds between strands
Why do you add primers during PCR?
• Attaches to / complementary to start of the gene / end of fragment;
• Replication of base sequence from here;
• Prevents strands annealing
Explain why ‘base-pairs’ is a suitable unit for measuring the length of a piece of DNA.
• DNA = 2 chains / joined by linking of 2 bases / A with T and G with C/ purine pairs with pyrimidine;
• Bases are a constant distance apart / nucleotides occupy constant distance/
• each base-pair is same length / sugar-phosphate is a constant distance;
Name one mutagenic agent.
• high energy radiation /ionising particles e.g. named particles/α, β, γ & X-rays;
• benzene;
• x rays/cosmic rays;
• uv (light);
• carcinogen / named carcinogen;
• mustard gas / phenols / tar (qualified);
A deletion mutation occurs in gene 1.
Describe how a deletion mutation alters the structure of a gene.
• removal of one or more bases/nucleotide;
• frameshift/(from point of mutation) base sequence change;
Describe the main stages in the copying, cutting and separation of the DNA.
• heat DNA to 95°C / 90°C;
• strands separate;
• cool so that primers bind to DNA;
• add DNA polymerase/nucleotides;
• use of restriction enzymes to cut DNA at specific base sequence/ breaks phosphodiester bonds
• use of electric current and agar/gel;
• shorter fragments move further;
Describe a plasmid
• circular DNA;
• separate from main bacterial DNA;
• contains only a few genes;
Suggest one reason why DNA replication stops in the polymerase chain reaction.
• Limited number of primers/nucleotides; /
Primers / nucleotides ‘used up’.
• DNA polymerase (eventually)denatures
Suggest why the restriction enzyme has cut the human DNA in many places but has cut the plasmid DNA only once.
• enzymes only cut DNA at specific base sequence/recognition site/specific point;
• sequence of bases/recognition site/specific point (on which enzyme acts)
• occurs once in plasmid and many times in human DNA;
• (max 1 if no reference to base sequence or recognition site)
Describe how the bacteria containing the insulin gene are used to obtain sufficient insulin for commercial use.
• use of fermenters;
• provides nutrients plus suitable conditions for optimum growth/named
• environmental factor;
• reproduction of bacteria;
• insulin accumulates and is extracted;
Explain what is meant by a vector.
• Carrier;
• DNA/gene; (context of foreign DNA)
• Into cell/other organism/host;
Explain how modified plasmids are made by genetic engineering and how the use of markers enable bacteria containing these plasmids to be detected.
• isolate TARGET gene/DNA from another organism/mRNA from
• cell/organism;
• using restriction endonuclease/restriction enzyme/reverse transcriptase to
• get DNA;
• produce sticky ends;
• use DNA ligase to join TARGET gene to plasmid;
• also include marker gene;
• example of marker e.g. antibiotic resistance;
• add plasmid to bacteria to grow (colonies);
• (replica) plate onto medium where the marker gene is expressed;
• bacteria/colonies not killed have antibiotic resistance gene and (probably) the TARGET gene;
• bacteria/colonies expressing the marker gene have the TARGET gene as well;
mRNA may be described as a polymer. Explain why.
• Made up of many (similar) molecules/monomers/nucleotides/units;
What is a DNA probe?
• (Short) single strand of DNA;
• Bases complementary (with DNA/allele/gene);
Name three techniques used by scientists to compare DNA sequences.
• Polymerase Chain Reaction
• DNA fingerprinting
• Gel electrophoresis
