The Control Of Gene Expression 8
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Define recombinant DNA What is the resulting organism known as What are the risks What is the way around this issue
DNA of two different species that has been combined Transgenic/Genetically modified organism(GMO) Infection+Rejection Modify own DNA/Multiply own DNA
Summarise the stages of DNA technology
-Isolate DNA that has the gene to code for the desired protein -Insertion of the isolated gene into a vector -Transformation of DNA into a suitable host -Identification of successful hosts -Cloning of successful host cells
What are the 3 methods to produce DNA fragments
-Converting mRNA into cDNA using reverse transcriptase -Using restriction endonucleodases to fragments of DNA -Creating the gene in a gene machine
GENE TECHNOLOGY Explain the process of using reverse transcriptase to form DNA fragments
• Cells that produce large amounts of mRNA for a required protein are identified • Cells are centrifuges to break open and mRNA is released • Free nucleotides are added and enzyme reverse transcriptase joins nucleotides with complementary bases together to form single stranded cDNA • More free nucleotides are added and DNA polymerase is added which forms double stranded DNA fragment
What is an advantage of using reverse transcription to form DNA fragments
The cDNA is intron free because it is based on the mRNA template
cDNA produced by reverse transcriptase doesn’t contain introns Why is this an advantage
Prokaryotic cells do not have the ability to remove introns from DNA
GENE TECHNOLOGY Describe the process of creating genes in a gene machine
• Sequence of amino acids for a desired protein is determined • complementary base triplets are worked out • Base sequence information is entered into a computer • The computer designs a series of small overlapping single strands of nucleotides called oligonucleotides • All the oligonucleotides are then joined to make a gene that contains no introns • Gene that is made is replicated using the polymerase chain reaction (PCR) • Gene is inserted into vector to allow Cloning to occur • Genes are checked using standard sequencing techniques and any with errors are rejected
What are restriction endonucleases?
Enzymes that cut up DNA at specific recognition sites
Where do restriction endonucleases naturally occur and why?
In bacteria, as a defence mechanism against viral DNA.
What are recognition sites?
Specific, palindromic DNA base sequences where restriction enzymes cut.
What does it mean if a recognition sequence is palindromic?
The sequence of bases reads the same on both strands in the 5’ → 3’ direction.
What are the two types of ends produced by restriction enzymes?
Sticky ends and blunt ends.
Why are sticky ends important in genetic engineering?
They allow DNA fragments from different sources to join together through complementary base pairing.
What do sticky ends do
They join two pieces of DNA by complementary binding
What are 2 ways DNA fragments can be cloned to amplify a sample
In vivo Cloning In vitro Cloning
What should be added to isolated DNA fragments to ensure transcription of these genes can occur
Promoter and terminator regions
What is the promoter region
Sequence of DNA that RNA polymerase binds to, to start transcription
What is the terminator region
Sequence of DNA that causes RNA polymerase to be released and ends transcription
What is a vector
Used to transfer genes from one organism to another
What is the most common type of vector used
Plasmid
INSERTION INTO VECTOR Why is the same restriction endonucleodase used to cut the DNA and plasmid
The dna fragments and plasmid has complementary sticky ends
After inserting the DNA fragment into the vector, what enzyme finishes it up and what does it do
DNA ligase catalyses condensation reaction to form phosphodiester bonds between nucleotides.
GENE TECHNOLOGY When the plasmid vector has been cut using an restriction endonucleodase, what 3 things can happen during Insertion of the gene in the vector
The complementary sticky ends of the plasmid and DNA fragment bind The plasmid recoils The DNA fragment recoils
GENE TECHNOLOGY Explain the process of transformation
Mix the bacteria cells with the recombinant plasmid in a medium containing calcium ions and increase the temperature (heat shocked) This increases the permeability of the cell membrane and allows the vector to enter the bacteria Cytoplasm