8B Genome Projects and Gene Technologies Flashcards
What is the genome?
Entire set of DNA, including genes in an organism
What did the 2003 human genome project do?
Mapped entire sequence of human genome
How do you sequence an entire genome?
Chop it up into smaller pieces and put back into order because methods only work on fragments of DNA
What does sequencing of simple organisms help identify?
Their proteins
What is the proteome?
All proteins made by an organism
Why is it easier to determine the proteome of simple organisms?
Have less non-coding DNA which makes it easier to find proteome from genome
Why does bacteria and viruses being simple organisms helpful in medical research?
Identify protein antigens and develop vaccine easier as less non-coding DNA
Why is it hard to translate the genome of complex organisms?
- Contain large sections of non-coding DNA
- Contain regulatory genes
What do regulatory genes do?
Determine when the genes that code for specific protein should be switched on or off
How have sequencing methods changed over time?
- Less expensive
- Large scale
- More automated
- Faster
What does recombinant DNA technology do?
Transfers a fragment of DNA from one organism to another
What are transgenic organisms?
Organisms that contain transferred DNA
Why can transferred DNA be used to produce a protein in cells of a recipient organism?
- DNA is universal
- Transcription and translation mechanisms are similar
What are the 3 ways a DNA fragment can be produced?
- Reverse transcriptase
- Restriction endonucleases (enzymes)
- Gene machine
How does reverse transcriptase produce a DNA fragment?
1) Extract and isolate mRNA from cell
2) T primers are added to the end of the mRNA
3) Reverse transcriptase is used to add free nucleotides to make a new strand of cDNA
4) New cDNA/mRNA strand is hydrolysed
5) DNA polymerase is added which synthesises double stranded DNA from the cDNA strand
How do restriction endonuclease (enzymes) produce a DNA fragment?
1) Restriction enzymes cut at specific palindromic sequences (recognition sequences)
2) When recognition sequences are present restriction enzymes cut the DNA fragment out by hydrolysis
3) The fragment cut can leave either blunt or sticky ends
4) Sticky ends can bind to a complimentary sticky end to form new DNA fragment
What are sticky ends?
Small tails of unpaired bases at each end of fragment after restriction enzymes have cut DNA
What is a palindrome sequence?
Antiparallel base pairs that reads the same in opposite directions
How does the gene machine produce a DNA fragment?
1) Sequence that is required is designed (if doesn’t already exist)
2) First nucleotide in sequence is fixed to some sort of support
3) Nucleotides are added step by step in correct order
4) Protecting groups make sure nucleotides join at right points to stop unwanted branching
5) Oligonucleotides (20 nucleotides long) are made and broken off from the support
6) Oligonucleotides are then joined together to make longer DNA fragments
What are the positives of using a gene machine?
- Quick and accurate
- Easily transcribed in prokaryotic cells
- No introns or exons
What are the 2 ways DNA can be amplified?
- In vivo amplification (vector)
- In vitro amplification (PCR)
What are the 3 main stages of in vivo amplification?
1 - DNA fragment inserted into a vector
2 - Vector transfers DNA fragment into host cell
3 - Identify the transformed host cells
How is the DNA fragment inserted into a vector (stage 1 of in vivo amplification)?
1) DNA fragment inserted into vector (plasmids or bacteriophages)
2) Vector cut by restriction enzymes so sticky ends are complementary to sticky ends on DNA fragment
3) Vector DNA and fragment DNA are mixed together with DNA ligases to form recombinant DNA (this is called ligation)
How is the recombinant DNA inserted into the host cell using a plasmid (stage 2 of in vivo amplification)?
1) Host cells have to be persuaded to take in plasmid vector and its DNA
2) The host cells are placed in ice-cold calcium chloride which makes them more permeable
3) Plasmids are added and mixture is heat-shocked (42 degrees) which encourages cell to take up plasmids
