Dna Technology Flashcards
What is recombinant DNA tech
Transfer of DNA from one organism to another
What is the first step in DNA tech
Isolate the gene required
This can be done through conversion of mRNA to complementary DNA using reverse transcriptase
Using restriction enzymes to cut fragment containing the desired gene from DNA
Creating the gene in a gene machine
How do you use reverse transcriptase in DNA tech
- mRNA is mixed with free DNA nucleotides and enzyme reverse transcriptase
- free DNA nucleotides bind to single stranded mRNA via complementary base pairing
- reverse transcriptase joins DNA nucleotides together to form a single stranded DNA strand copy of mRNA template called cDNA
- the addition of further DNA nucleotides and DNA polymerase is then used to make cDNA double stranded
What are the advantages of using reverse transcriptase
- cells contain many mRNA for genes they are expressing the gene ( many copies )
- using mRNA means introns have been removed ( prokaryotes can’t express introns )
What are the disadvantages of reverse transcriptase
mRNA does not contain promoter and terminator regions of the target DNA sequence, so these have to be added to any cDNA produced before the gene can be expressed during in vivo techniques
What are restriction endonuclease
Enzymes which cut DNA at specific base sequences ( recognition sequences )
Different restriction endonucleases cut dna at different specific recognition sites as the shape of the site is complementary to the enzymes active site
What does palindromic mean
If recognition sequences are palindromic the base pairs read the same in the opposite direction
What does restriction endinuclease produce
Blunt ends - straight
Sticky ends - staggered
What is a gene machine
Artificial gene synthesis - making a gene in a lab by entering the desired sequence of nucleotide bases into a computer
What are oligonucleotides
Small, overlapping single strands of nucleotides that can be assembled into the desired gene
Made by computers with a small fragment of genes
How does a gene machine work
1) bio informatics - desired nucleotide sequence fed into a computer
2) synthesis of oglionucleotides - by adding nucleotides one at a time
3) assembly of gene - oligonucleotides are overlapped then joined together and made double stranded using pcr
4) gene cloning - gene is inserted into bacterial plasmid
5) genes are sequenced and those with errors are rejected
What is an advantage of using a gene machine
As the dna is usually worked out from the protein structure, it does not contain any introns
Therefore these artificial genes are readily transcribed and translated by prokaryotes that also do not have non coding areas of their dna
Quicker
Highly accurate
What is in Vivo cloning
Copies of dna fragments made within a living organism
Foreign fragment placed into organisms dna as it divides, replicates foreign dna along with its own
Steps of in vivo cloning
1) isolation of gene and addition of promoter and terminator regions
2) insertion of dna fragment into vector
3) transformation
4) identification
What occurs in step 1 of in vivo cloning
Isolate gene
Add promoter and terminator regions to produce protein
Promoter dna sequences are regions help rna polymerase bind to the target gene for transcription producing mRNA
Terminator regions tell rna polymerase we’re to stop transcription
What occurs in step 2 of in vivo cloning
Dna fragment placed into the vector by cutting it open using the same restriction endonuclease that was used to isolated the dna fragment
Produces complementary sticky ends between ends of dna fragment and cut ends of vector dna
Dna fragments joins to vector by complementary base pairing
Dna ligase joins them together with a phosphodiester bond
This is recombinant dna
What occurs in step 3 of in vivo cloning
The dna fragement in the vector is transferred to the host cell
Method differs depending on vector used
- plasmid vectors : circular pieces of dna found in bacterial cells. If bacterial cells are placed in solution of recombinant plasmids they can be encouraged to take up the plasmids from the solution under certain conditions
- host cells which take up recombinant dna are referred to as transgenic organisms
What occurs during step 4 of in vivo cloning
Use of markerbgenes to detect genetically modified cells or organisms
Using marker genes
- antiobotic resistance gene - marker gene codes for antibiotic resistance when cells are grown in the presence of this antibiotic only transformed organisms will grow
- flourensence gene - marker gene codes for flouresemce when uv light is shone on cells, the transformed organisms will glow
What is a marker gene
Gene inserted into the vector which gives the transformed organism a specific characteristic that can be used to identify it
What occurs in step 5 of in vivo cloning.
The identified bacteria are cultured on a large scale in a fermenter, they make the new protein and it is extracted and purified
What occurs during in vitro cloning
The polymerase chain reaction ( PCR)
What is PCR
Takes samples of viral DNA and amplifies it. A reaction mixture is set up containing the dna sample, free dna nucleotides, primers and heat resistant dna polymerase Automated using PCR thermal cycling machine
What are primers
short pieces of single stranded dna with complementary base sequences to bases at the start of dna fragment you want to isolate
What occurs in the PCR
1) denaturation. - heated to 94 degrees to break H bonds between 2 strands of DNA for 30secs. 2) annealing - cooled to 50 -60 degrees so that primers can anneal to the single strand for 30 secs. 3) extension - heated to 74 degrees to allow heat resistance dna polymerase to work
