Recombinant DNA Flashcards
(24 cards)
What is recombinant DNA
Term used to describe the combination of two DNA strands that are constructed artificially
What does rDNA produce?
Recombinant proteins
Procedure of creating rDNA
1) choosing the appropriate host organism and cloning vector
2) prepararion of vector DNA and DNA to be cloned
3) creation of rDNA
4) introduction of rDNA to host organism
5) screening for rDNA with soecific properties sought from host irganisms
Producing rDNA detailed
1) cooy DNA (extract it form any cell)
2) select the genes (northern or southern blotting techniques)
3) detection (chemoluminiscent)
4) PCR
What is a blotting tecnique
A tecnique that allows you to copy the gene by running the sequence over a gel and then pasting it on a membrane
Steps in southern blotting
1) gel electrophoresis soarates dna fragments produced by restriction enzymes
2) bands on the gel are immobilized by blottiyonto nitrocellulose paper
3) radioactive probes bind to fragments with complementary base sequences
Differences of southern and northern blotting
Northern blot is for RNA mixtures and is only for single stran rna
Southern blot is for DNA
What does pcr allows us to do?
Allows us to amplify it or make millions of copies of the gene
Enzyme needed in PCR
Taq polymerase
Who invented PCR
Kari mulles
Steps in PCR
1) denaturation
2) annealing
3) extension
Bacteria were we isolate taq polymerase
Thermus aquaticus
Detailed PCR steps
1) denaturation: highest temperature (96 C) separationcof DNA (single strands now)
2) cool the reaction so the primers ass complementary bases (55-65 C)
3) taq polymerase extends the chain (72 C)
What do you do after PCR?
Electrophoresis to make sure you got the gene you wanted
What happens in every cycle of PCR?
We double the amount of DNA
2,4,8,16,32,64……
How do you build a DNA sequence?
With machines known as DNA synthesizers
What do scientists do for the cell to replicate rDNA
They join rDNA to another piece of DNA containing a replication “start” signal. This way whenever the cell copies its own dna it also copies rDNA
What are plasmids
Small circular dna molecules bacteria contain
What is restriction enzyme
Enzyme that cuts dna strands at specific sequences creating sticky ends. Then dna ligase join the two sticky ends
What is a genetic marker
A gene that makes it possible to distinguish bacteria that carry the plasmid form those that don’t
What are the vectors of rDNA
Lambda and plasmid
Differences of rDNA vectors
Lambda is viral and can carry more rDNA than the plasmid
Plasmid is with bacteria and is easier to use
Describe the process scientist use to copy DNA
PCR
How do scientists use rDNA?
They attach it to another dna which makes it possible to change the genetic composition of living organisms
