Gene technologies Flashcards
Define proteome
All the proteins produced in a given type of cell or organism at a given time
Define recombinant DNA
DNA from two different sources joined together
Define in vivo
Experiments performed within the living organism
Define in vitro
Experiments performed test tubes/ petri dishes outside of the living organism
Describe how using restriction enzymes to cut a fragment containing desired gene from DNA ( Step 1 - Isolation)
. Restriction endonuclease are enzymes which cut DNA at specific bas sequences
. Different restriction endonuclease cut DNA at different specific base sequences because the shape of the recognition site is complementary to the enzymes active site
. These recognition sites are palindromic
. If recognition site for these enzymes are either side of the DNA fragment you want, you can use restriction endonuclease to cut it out
Define sticky ends
Short, single standard section at then end of a DNA molecule, the result of staggered cutting with a restriction endonuclease. Sticky ends can be used to bind DNA fragments together, if another DNA molecule has complementary sticky ends
Define restriction endonuclease
Cut DNA at specific base sequences/ recognition sites. Breaks phosphodiester bonds. Produces sticky ends
Define DNA ligase
Ligase joins DNA and plasmids/ vectors. Joins stick ends together. Reforms phosphodiester bonds
Define recognition sites
. 4 to 8 base pairs long nucleotide sequence. Where restriction enzyme attaches and cuts
Define palindrome
Base pairs reads the same in opposite directions
Describe the conversion of mRNA to cDNA, using reverse transcriptase ( step 1 - isolation)
. Beta cells form islets of Langerhans in the human pancrease, where the insulin gene is transcribed into mRNA
. mRNA coding for insulin is extracted from the cell
. mRNA coding for insulin
. mRNA acts as a template on which cDNA strand is formed using reverse transcripts
. Single stranded cDNA
. New DNA nucleotides form hydrogen bonds, with complementary base pairs. Adenine to Thymine and Cytosine to Guanine
. DNA polymerase joins the new adjacent nucleotides / forms phosphodiester bonds and double stranded cDNA Is formed
. Copy of human insulin gene can now be inserted into host cells/ cloned
What are the advantages of using reverse transcriptase
. mRNA is easy to obtain as it can be found outside of the nucleus
. Using mRNA isolated from Cytoplasm means intros have been removed
Describe creating the gene in a gene machine ( step 1- isolation)
. Protein sequence analysis
. Amino acid sequence worked out
. mRNA sequence worked out
. DNA nucleotide base sequence worked out
. DNA nucleotide base sequence is uploaded into the computer
. Computer produces DNA base sequence by producing overlapping single strands of nucleotides called Oligonucleotides which can then be assembled into the gene
What are the advantages of using the gene machine
. Faster to use the gene machine rather the using reverse transcriptase or restriction endonuclease
. There are more steps involved in isolation the gene/ mRNA as well as the enzyme catalysed reactions
. Highly accurate
Define vector
A DNA carrier used to transfer DNA into host cells / organisms
Define promoter region
Binding site for RNA polymerase on DNA
Define RNA polymerase
Enzyme that joins RNA nucleotides to form mRNA during transcription
Define transcription factors
Protein that binds to promoter region and RNA polymerase to begin transcription
Define terminator region
Region of DNA that releases RNA polymerase to stop transcription
Describe inserting the gene of interest into a vector ( step 2 )
. The most common type of vector is a plasmid which can be inserted into bacteria
. To prepare the vector for the gene of intreset the vector has to be opened by using the same restriction enzyme
. So the sticky ends are complementary
. Dna ligase is then used to recombine the 2 pieces if DNA
. This is known as recombinant DNA
Describe chemical transformation ( part 1 of step 3)
Positive charge attracted to negative charge so DNA will not be repelled by membrane
. DNA has a negative charge
. Ca 2+ has a positive charge
Describe heat shock ( part 2 of step 3 - transformation)
. Drop temperature- less kinetic energy/ less vibrations
. Quickly increase temperature- burst of kinetic energy/ lots of vibrations
. Creates gaps in membrane for DNA to move into the bacteria cell
Describe step 4 : Identification of the transformed bacteria
. Some of the plasmids will have resealed without becoming recombinant- we don’t want this
. Some of the gene of interest will have resealed without becoming recombinant- we don’t want this
. There will be some recombinant DNA - we want this
. Some of the bacteria will not have transformed at all- we don’t want this`
Define antibiotic resistance
The ability for microorganisms to withstand the adverse affect of antibiotics
Define gene marker
Is a gene or DNA sequence with a known location on a chromosome ( plasmids) that can be used for identification. Can be modified to be fluorescent or radioactive
Describe the procedure for PCR ( Polymerase chain reaction)
1) Separation of DNA strand- The mixture is heated to 95c which breaks hydrogen bonds between the bases, therefore separates the strands and produces single stranded DNA molecules = denatured
2) Addition of the primers- The temperature is reduced to 55-60c to allow primers to bind to the ends of the single stranded DNA molecules= Annealing
3) Synthesis of DNA- The temperature is increased to 72c. This enables the free DNA nucleotides to attach by complementary base pairing. The new DNA strands are built using the enzyme Taq polymerase which joins the phosphodiester bonds= extension. The first cycle is complete
4) The cycle is repeated
5) The 2 resulting DNA molecules make up the template DNA for the next cycle, thus amplifying the amount of DNA duplicated for each new cycle
What are the pros and cons using in vitro (PCR)
Pros
- Doesn’t require a vector
- Doesn’t require living cells/ Bacteria
- Very fast : hours to produce large quantities
Cons
- Must know sequence for primers to bind- must be studied before
- Less accurate, any error will be copied in subsequent cycles
- Can only accurately copy shorter sequences of bases- about 1000 base pairs
Pros and cons using in vivo ( recombinant DNA )
Pros
- More accurate, Mutations are very rare so less errors
- Can copy unknown sequences that have not been studied before
- Can accurately copy longer sequences of bases, about 2 million base pairs
Cons
- Require a vector, viruses may have harmful side effects
- Require living cells/ bacteria
- Slower, days or weeks to produce large quantities
Uses of PCR
. Forensic analysis- DNA available from crime scenes
. Screening- DNA from embryo can be amplified for genetic testing
. Tissue typing
. Tissue analysis from endangered/ extinct animal samples
. Paternity testing
. Identification of human remains
Describe Gel electrophoresis
. DNA is hydrolysed by restriction endonuclease
. Hydrolysed DNA placed in well at one end of an agarose gel
. Gel is placed in a buffer
. Electrical current is passed through the gel
. DNA is negatively charged ( due to phosphate group)
. DNA fragments migrate towards the positively charged end of the gel
. Smaller DNA fragments migrate through the gel more quickly/ further than larger fragments
Describe the visualising the DNA fragments: Southern Blotting and autoradiography
. Immerse gel in alkaline solution so two strands of DNA separated/ make DNA single stranded
. Southern blotting- cover with nylon/ absorbent paper ( to absorb DNA)
. DNA fixed to nylon/ membrane using UV light
. Add radioactive or fluorescent probe to single stranded DNA
. Show up using exposed to X-rays or UV light
Define DNA hybridisation
. Two different strands of DNA/ RNA anneal/ join / bind together
. e.g. DNA + Probe = hybridisation
. Used to identify complementary alleles or how similar species are
Define DNA probe
. Short single stranded of DNA ( 8-12 base pairs long)
. Bases complementary with DNA/ allele/ VNTR
Describe how are DNA probes used to identify alleles or genes
- DNA extracted from sample
- Amplified DNA using PCR
- DNA hydrolysed into fragments using restriction endonuclease
- Must leave alleles that is being screened for intact
- DNA fragments separated by size using gel electrophoresis
- Mixture put into wells on gel and electric current passed through
- Immerse gel in alkaline solution so two strands of DNA separated as hydrogen bonds break
- Southern blotting- cover with nylon/ absorbent paper to absorb DNA
- DNA fixed to nylon membrane using UV light
- Radioactive/ florescent marker/ probe added which is complementary to allele
- Excess probes are washed away
- Radioactive probe- identified using x-ray film/ autoradiography or florescent probe- identified using UV light
Describe how to produce genetic fingerprints ( VNTR)
- DNA extracted from sample
- Amplify DNA using PCR
- DNA hydrolysed into fragments using restriction endonuclease
- Must leave VNTR that is being screened for intact
- DNA fragments separated by size using gel electrophoresis
- Mixture put into wells on gel and electric current passed through
. Immerse gel in alkaline solution so two strands of DNA separated as hydrogen bonds break - Southern blotting- cover with nylon/ absorbent paper to absorb DNA
- DNA fixed to nylon membrane using UV light
- Radioactive/ florescent marker/ probe added which is complementary to VNTR
- Excess probes are washed away
- Radioactive probe- identified using x-ray film/ autoradiography or florescent probe- identified using UV light
Uses of genetic fingerprinting
. Forensic science
. Determine genetic relationships
. Medical diagnosis
. Determining genetic variability in a population
. Animal and plant breeding
Genetic fingerprinting and VNTR’S
. VNTR ( variable number tandem repeats) are repetitive sequences of non-coding DNA
. Can vary by:
- Number of bases in the repeated sequence varies
- Number of times the sequence is repeated
. Difference in VNTR can occur due to mutation and are retained as in the non-coding DNA
. Restriction endonuclease cut at recognition sites on either side of the VNTR
. Distance between recognition sites vary and are unique to each person
. Produces different sized fragments
What are the benefits with recombinant DNA technology in agriculture with example
. Higher yield
. More nutritious crops
. Reduces famine and malnutrition
. Drought resistant crops
. Golden rice , gene from soil bacteria and maize, high in beta-carotene makes vitamin A in humans. Vitamin A deficiently leads to blindness
What are the concerns with recombinant DNA technology in agriculture
.Mono-culture- all crops susceptible to the same diseases and reduced biodiversity
. Weeds become resistant to herbicides if transformed crops interbreed with wild crops
What are the benefits with recombinant DNA technology in Industry
. Enzymes made in large quantities, for less money by transformed bacteria
. Chymosin used in cheese making
What are the concerns with recombinant DNA technology in Industry
. Purification of proteins may introduce toxins into the food industry
. Labelling may not be clear so people may eat food made from transformed organisms
. Large biotech companies using transformed organisms out complete smaller companies not using them
What are the benefits with recombinant DNA technology in medicine
. Drugs and vaccines made from transformed organisms quickly, cheaply and in large quantities
. Instead of using pig insulin, human insulin made using cloned human gene
What are the concern with recombinant DNA technology in medcine
. Some big tech companies own the patent on particular GM seeds, may charge higher prices
. Could make designer babies
. Debate over who owns human genetic material once it has been removed from the body
. Non GM crops may be contaminated by GM crops
Define gene therapy
The treatment of genetic diseases by giving patients healthy copies of defective genes. There are two main types of gene therapy: Somatic ( body cells) and Germline (Sex cells/ gametes)
Describe somatic gene therapy
. Functional gene inserted into body cells
. Vectors required
. If successful- only target cells are recombinant
. Functional gene not passed onto child
Describe germline gene therapy
. Functional gene inserted into sperm/egg
. Vector would be needed but currently illegal so not tested
. If successful- all cells are recombinant
. Functional gene will be passed down to offspring
Effectiveness and risks of gene therapy
. Short term treatment
. Treatment must be separated
. Difficult to get allele into the DNA in a functional state
. Long term effects unknown
. Vectors/ viruses cause harmful side effects
. Modified cells/ vectors cause immune response/ rejection
