Recombinant Gene Tech Flashcards
Outline the importance of genome sequencing projects:
Genome sequencing can be used to link specific gene mutations to genetic diseases.
Describe how to determine the genome and proteome of simple/complex organisms:
The proteome is all of the proteins produce by a cell
In simple organisms, the genome codes for the proteome as no non-coding regions or regulatory eenes are present.
The majority of the genome of the cell may not code for their proteome due to large non-coding regions in complex organisms.
Can you explain how reverse transcriptase can be used to isolate a gene?
mRNA is mixed with free DNA nucleotides and reverse transcriptase.
Complementary base pairing
Reverse transcriptase joins DNA nucleotides together to form a DNA strand complementary to mRNA (cDNA).
Addition of further DNA nucleotides and DNA polymerase used to make cDNA double stranded.
Can you explain the main stages of DNA technology?
Identification
isolation Restriction/Reverse
Multiplication PCR/Vivo
Transfer Vector
Identification Marker
Can you explain how restriction endonucleases can be used to isolate a gene?
Restriction endonucleases hydrolyse DNA at specific recognition base sequences.
Specific Restriction endonucleases cut at specific recognition base sequences because the shape of the site is complementary to the enzymes active site.
Produces Blunt or Sticky ends specific to the restriction enzyme.
The recognition sequences are palindromic.
N.B Recog seq cannot occur within the DNA fragment you want to isolate as this would hydrolyse the fragment prematurely.
Can you explain how a gene machine can be used to manufacture a gene?
Desired nucleotide sequence fed into computer
Oligonucleotides synthesised
Gene assembled, Oligonucleotides are overlapped, joined together and made double stranded using PCR.
Gene cloning by insertion into a bacterial plasmid.
Genes are sequences and those with errors are rejected.
Can you explain why sticky ends are important and how a DNA fragment can be inserted into a vector?
Sticky ends can be used to anneal DNA fragments together if the other DNA molecule has complementary sticky ends (cut by same enzyme)
Can you explain how the DNA of the vector is introduced into host cells?
In vivo cloning
Promoter and terminator regions added to DNA fragment.
Inserted into vector with restriction endonucleases and ligases.
Restriction endonuclease Cuts open the vector DNA using the same Restriction used to isolate the DNA fragment.
Producing complementary sticky ends for the fragment to comp base pair and join together with DNA ligase which catalyses the condensation reaction to join the sugar phosphate backbone of the fragment and vector DNA.
Recombinant DNA is transferred into host cell, creating transgenic organism.
Markers can be used to detect GM
Can you explain what gene markers are and how they work?
gene inserted into the vector which gives the transformed organism a specific characteristic that can be used to identify it.
Genes that are replaced by recombinant DNA can also be tested for such as disrupting an Ab resistance gene when inserting the DNA fragment.
Can use replica plating.
How would you prepare the DNA fragment for insertion?
Describe the polymerase chain reaction and what the process requires:
Polymerase chain reaction (PCR) make millions of copies of a fragment of DNA.
A reaction mixture is set up containing the DNA sample, free DNA nucleotides, primers and heat resistant DNA polymerase.
Explain how the polymerase chain reaction is carried out:
Denaturation: Heated to 94 °C to break hydrogen bonds between two strands of DNA for 30s. (1)
Annealing: Cooled to 50 to 60°C so that primers can anneal (bind) to the single strands for 30s. (2)
Extension: Heated to 74°C - temperature that heat resistance DNA polymerase works. (3)
T.aq (DNA) polymerase joins adjacent DNA nucleotides together in condensation reactions forming phosphodiester bonds. This forms a newly synthesised complementary strand for required section of DNA only.
Two new copies the DNA fragment are formed, one cycle complete.
The cycle repeats, DNA is heated to 94°C, and this time four strands separate.
This can be repeated many times, the number of copies of DNA fragments doubles with each cycle. (4).
You can calculate the number of DNA molecules produced per cycle by using the formula:
2n
where n = number of cycles
Explain the advantages and disadvantages of in vivo cloning:
‘In vivo’ cloning
Can be used to produce protein or mRNA from the inserted DNA as well as the target DNA.
Can be used to clone large DNA fragments i.e. up to 2 Mb long.
Slower – One DNA replication per cell division, limited by growth of host cell.
DNA, RNA and proteins produced are modified – i.e. introns removed, groups added by cell.
Need to isolate DNA fragment from DNA before it can be inserted into the host cell.
Cannot be used to copy partly broken down DNA.
Not very sensitive – need large DNA sample to start with.
Cells have mechanisms for correcting errors that are made when copying genes. This makes ‘in vivo’ copying more accurate.
Explain the advantages and disadvantages of in vitro cloning:
Can only be used to copy DNA.
Cloning becomes unreliable when used to copy DNA fragments longer than 1000 base pairs (1kbp).
Quicker – can produce millions of copies of target DNA within hours.
No modification of DNA.
Only replicates target DNA, no need to isolate DNA fragment from DNA.
Can be used to copy partly broken down DNA.
Very sensitive – only requires a small DNA sample to start with.
Lacks error-correcting mechanisms, so the error rate is higher than cell-based methods.
