8.4 Gene technologies Flashcards
(14 cards)
What is recombinant DNA technology
Transfer of DNA fragments from one organism or species, to another.
Explain why transferred DNA can be translated within cells of the recipient (transgenic) organism
- genetic code is universal
- transcription and translation mechanisms are universal
Describe how DNA fragments can be produced using restriction enzymes
- restriction enzymes cut DNA at specific base “recognition sequences” on either side of the desired gene
- many cut in a staggered fashion forming “sticky ends” - single stranded overhang
Describe how DNA fragments can be produced by mRNA
- isolated mRNA from a cell that readily synthesis the protein coded for by the desired gene
- mix mRNA with DNA nucleotides and reverse transcriptase - reverse transcriptase uses mRNA as a template to synthesise a single strand of complementary DNA (cDNA)
- DNA polymerase can form a second strand of DNA using cDNA as a template
Suggest two advantages of obtaining genes from mRNA rather than from the DNA removed from cells
- much more mRNA in cells making the protein than DNA - easily extracted
- in mRNA. introns have been removed by splicing (in eukaryotes) whereas DNA contains introns
(so can be transcribed and translated by prokaryotes who can’t remove introns by splicing)
Describe how DNA fragments can be produced using a gene machine.
1. synthesised fragments of DNA quickly and accurately from starch without the need for a DNA template
- amino acid sequence of protein determined, allowing base sequence to be established
- these do not contain introns so can be transcribed and translated by prokaryotes
Name an in vitro and in vivo technique used to amplify DNA fragments
1. in vitro (outside a living organism) - polymerase chain reaction (PCR)
2. in vivo (inside a living organism)
- culturing transformed host cells e.g. bacteria
Explain how DNA fragments can be amplified by PCR
reaction mixture: DNA fragment, DNA polymerase (e.g. taq polymerase, primers and DNA nucleotides)
- mixture heated to 95C
- this separates DNA strands
- breaking H bonds between bases - mixture cooled to 55C
- this allows primers to bind to DNA fragment template strand
- by forming hydrogen bonds between complementary bases - mixture heated to 72C
- nucleotides align next to complementary exposed bases
- DNA polymerase joins adjacent DNA nucleotides, forming phosphodiester bonds
PCR
step1. mixture heated to 95C
- this separates DNA strands
- breaking H bonds between bases
PCR
step2. mixture cooled to 55C
- this allows primers to bind to DNA fragment template strand
- by forming hydrogen bonds between complementary bases
PCR
step3. mixture heated to 72C
- nucleotides align next to complementary exposed bases
- DNA polymerase joins adjacent DNA nucleotides, forming phosphodiester bonds
The PCR cycle is ___
In each cycle, the amount of DNA __
The cycle is repeated
in every cycle, the amount of DNA doubles causing an exponential increase (2^n)
Explain the role of primers in PCR
- primers are short, single-stranded DNA fragments
- complementary to DNA base sequence at edges of regions to be copied/start of desired genes
- allowing DNA polymerase to bind to start synthesis (can only add nucleotides onto pre-existing 3’ end)
- two different primers (forward and reverse) are required (as base sequences at ends are different)
Suggest one reason why DNA replication eventually stops in PCR
there are limited number of primers and nucleotides which are eventually used upS
Summarize the steps involved in amplifying DNA fragments in vivo
- add promoter and terminator regions to DNA fragments
- insert DNA fragments and marker genes into vectors (e.g. plasmids) suing restrictions enzymes and ligases
- transform host cells (e.g. bacteria) by inserting these vectors
- detect genetically modified (GM)/transformed cells/organisms by identifying those containing the marker gene (e.g. that codes for a fluorescent protein)
- culture these transformed host cells, allowing them to divide and form clones
following this, DNA can be extracted from the host cells if needed or the host cells can produce a protein coded for by a gene in the DNA fragment
