22: Gene Technology Flashcards
What are the 3 ways of obtaining DNA fragments?
- using enzymes, restriction endonucleases
- using a gene machine
- conversion of mRNA into cDNA (complementary DNA) via the enzyme reverse transcriptase
Describe obtaining DNA Fragments via Restriction Endonucleases:
- group of enzymes which naturally occur in bacteria
- they cut DNA at a specific sequence of bases “recognition sequences”
- this occurs by the hydrolysis of phosphodiester bonds in the sugar-phosphate backbone
- the “cuts” occur between opposite bases (within the 2 strands) in a staggered fashion to create sticky ends
Define Sticky Ends:
What’s the relationship between recognition sequences and sticky ends?
- single-stranded section of DNA that form an overhang at the end of double-stranded molecules
- they are palindromic, meaning that they have the same base sequences but in reverse
Describe obtaining DNA Fragments via the “Gene Machine”:
- the amino acid sequence is determined
- the mRNA codons for each amino acid are looked up
- from this, the complementary DNA triplets are worked out, leading to the production of the gene
- genes are then checked for biosafety + biosecurity
Give 2 Advantages of the “Gene Machine”:
- sequence of bases are produced rapidly + accurately
- genes can be free of introns and other non-coding DNA, (so they can be transcribed + translated by prokaryotic cells)
Describe obtaining DNA Fragments via “reverse transcriptase”:
- sometimes RNA from viruses needs to be converted into DNA
- the enzyme reverse transcriptase is used to convert mRNA to cDNA (complementary DNA)
Give 3 Advantages of using “reverse transcriptase”:
- cDNA produced would have introns removed (due to it being made from mRNA), so can be used in prokaryotes
- only a few types of mRNA in each cell depending on the gene that is expressed, compared to the huge number of DNA sequences for genes
- mRNA is at a high concentration within cells, therefore easier to isolate compared to a gene, which has 2 copies of homologous chromosomes
What is the second step of Gene Technology?
What equipment is required?
- amplification of DNA (PCR)
- primers, thermostable DNA polymerase, thermal cycler, free nucleotides, DNA fragment
What is a primer?
- short sequence of single-stranded nucleotides with a specific base sequence
Describe the Polymerase Chain Reaction:
- temperature is raised to 95 degrees, breaking the hydrogen bonds to separate the 2 strands of DNA
- temperature is then decreased to 55 degrees, to allow primers (single-stranded nucleotides) to attach to the complementary base sequences at the ends of the newly formed 2 strands
- thermostable DNA polymerase is added as the temperature is increased to 72, as this is the optimum temperature
- therefore, the free DNA nucleotides attach to the single stranded templates via complementary base pairing
- DNA polymerase joins free adjacent nucleotides together via the formation of phosphodiester bonds
Give 3 advantages of PCR:
- automated
- rapid
- doesn’t require living cells (so faster)
What is Gel Electrophoresis used for?
- used to separate different pieces of DNA on the basis of length from the DNA sample
Describe Gel Electrophoresis:
- DNA samples are amplified via PCR, and then cut into fragments via restriction enzymes
- fragments placed into wells at one end of a thin slab of gel
- electric current passed through the well, causing the DNA to move towards the positive electrode (due to phosphate group)
- the smaller the length of the DNA molecule, the further down the gel it moves
What is step 3 of Gene Technology?
What are gene probes?
Following Gene Electrophoresis
Genetic Screening + Diagnosis
- gene probe: single stranded, short sequence of DNA nucleotides with a complementary base sequence to the gene/allele being screened for
Describe the process of Gene Probes:
- DNA sample amplifies DNA containing potential gene/allele
- this is then cut into fragments via restriction endonucleases
- fragments are then separated by length + charge via gel electrophoresis
- gene probe is added with label (containing fluorescent/radioactive)
- if present the gene probe binds to the allele/gene allowing for identification
What is step 4 of Gene Technology?
Describe this process:
What are VNTRs?
genetic fingerprinting:
- diagnostic tool used to analyse small differences in non-coding DNA between genes, between individuals of the same species
- focuses on VNTRs (Variable Number Tandem Repeats)
- non-coding sections of unique DNA in between genes
What is the correlation between VNTR similarity and heritence?
- the more genetically linked, the more similar the VNTRs
Describe the Overall Process of Genetic Fingerprinting:
(step 1-4)
- Extraction: DNA extracted from samples containing nucleus, and is amplified via PCR
- Digestion: restriction endonucleases are used to cut recognition sites close to, but not within, VNTR sequences within non-coding DNA
- Separation:
- gel electrophoresis used to separate DNA fragments via length and charge
- these separated fragments are then added to alkali to become single-stranded - Binding of DNA probes:
- radioactive/fluorescent DNA probes added, and would bind to complementary base sequences in VNTRs if present - Visualisation:
- if radioactive DNA probe is used, X-ray film is placed over the gel
- if fluorescent probe used, sample is exposed to UV light, and then an image is taken
Give 4 uses of Genetic Fingerprinting:
- to determine paternity:
- individuals inherit half of genetic material from mother + half from father
- each band of DNA fingerprint should have corresponding band in the one of the parents DNA fingerprint - forensic science:
- DNA left at crime scene allows to determine who was present at the scene of crime - Medical Diagnosis:
- sample of DNA from a person with a disease, can be compared to those showing symptoms/or genetically related - Plant + Animal Breeding:
- genetic fingerprinting can prevent interbreeding
Why can Recombinant DNA technology work across organisms?
What is In Vivo Cloning?
What steps are these after?
- genetic code is universal, and so it transcription + translation mechanisms
- genes cloned for in cells
- 1 + 2 (DNA fragment formation + amplification)
What are the 5 steps of In-Vivo Cloning?
- Preparation of the DNA fragment for insertion
- Insertion of the DNA fragment into a vector
- Transformation
- Identification of transformed cells using marker genes
- Growth on large scale
Describe the 1st step of In-Vivo Cloning:
Preparation of DNA fragments for insertion:
- involves the addition of extra lengths of DNA:
1. promotor sequence added to the start to provide a binding site for RNA polymerase
2. terminator sequence at the end to release RNA polymerase at the end of transcription
Describe the 2nd step of In-Vivo Cloning:
Insertion of DNA fragment into a vector:
1. a restriction enzyme is used to cut the gene from DNA, to create sticky ends
2. the same restriction enzyme is then use to cut the plasmid to create complementary sticky ends
3. the gene + plasmid are then mixed into a tube, and they join due to the complementary sticky ends formed from the same restriction enzyme
4. DNA ligase is added to the mixture to join the sticky ends together by forming phosphodiester bonds
5. the plasmid is now a recombinant plasmid
What is a vector?
What is a plasmid used for?
Why does the same restriction enzyme need to be used?
- carrier used to transport DNA into a host cell
- to transfer DNA into bacterial cells
- so the sticky ends are complementary to allow the recombinant plasmid to be formed
