3.8.4.1 Recombinant DNA Technology Flashcards
What is recombinant DNA technology?
A form of genetic modification
What does transgenic mean?
Genetically modified
What is isolation?
The isolation of the DNA fragments that have the gene for the desired protein
What is insertion?
The insertion of the DNA fragment into a vector
What is transformation?
The transfer of DNA into suitable cells
What is identification?
The identification of the host cells that have successfully taken up the gene by use of the gene markers
What are the steps of recombinant gene technology?
Isolation Insertion Transformation Identification Growth / cloning
What are the methods of producing gene fragments?
Conversion of mRNA to cDNA using reverse transcriptase
Using restriction endonucleases to cut fragments containing the desired gene from DNA
Creating the gene in a gene machine
What do endonucleases do?
They break up nucleotides
What is a blunt end?
When DNA is cut straight through the double strand without seperating bases
What is a sticky end?
When DNA is cut in a staggered fashion This usually happens when the code on one strand is pallindromic e.g. A G C G C T T C G C G A
What is a DNA fragment?
The DNA segment between the two cuts
What are the sticky ends on the fragment complimemtary to?
The sticky ends on the plasmid
What is the proces of using reverse transcriptase?
Start with the mRNA code
The reverse transcriptase forms a complimentary strand of DNA, called cDNA
Single-stranded cDNA is isolated by hydrolysis of the mRNA with an enzyme
Double-stranded DNA is formed on the template of the cDNA using DNA polymerase
What does reverse transcriptase produce?
DNA with no noncoding DNA
What is the process of using restriction endonucleases?
Each type of these enzymes cuts a DNA double strand at a specific sequence of bases
This leaves two blunt ends
Others cut in a staggard fashion
What do restriction endonucleases produce?
cDNA (noncoding DNA)
What is the process of using a ‘gene machine’?
You need to work out the amino acid sequence first
This means you should know the mRNA codons and therefore the DNA triplets
The desired sequence of nucleotide bases is fed into a computer
The computer designs oligonucleotides
Oligonucleotides are short pieces of single-stranded DNA which are joined together to make a gene
It is quicker to create smaller strands then join everything together, base by base
The polymerase chain reaction constructs the complementary strand
What is the process of insertion?
DNA ligase joins the DNA fragment and vector (ligation)
This forms recombinant DNA
A promotor region is a section of DNA at the end of the gene where DNA polymerase binds to
The nucleotide bases of the promotor attach both RNA polymerase and transcription factors, and so begin the process of transcription
Attaching the promotor is essential for transcription
A terminator must also be added to stop transcription at the appropriate point
At this point you have a recombinant plasmid
If you use restriction enzymes, introns are present and splicing is required
What is a vector?
A vector carrier DNA into a host cell
What does using a gene machine produce?
Coding DNA