Chapter 21 Flashcards
(45 cards)
Explain what a DNA fragment is?
small pieces of DNA
we can isolate them and insert them into cells to produce large amounts of desired proteins
What 2 ways can enzymes be used to produce DNA fragments?
- RNA to DNA
- cut DNA to specific sequence or use gene machine
Explain what recombinant DNA is?
- recombinant DNA technology involves the transfer of fragments of DNA from one organism or species to another
What are the 5 stages of the process of making a protein by recombinant DNA technology
- Isolation - of DNA that codes for proteins
- Insertion - of DNA into a vector
- Transformation - of DNA into a host
- Identification - of successful hosts by gene markers
- Growth/cloning - of host cells
What are the 3 methods of isolation of DNA fragments
- using reverse transcriptase to convert mRNA to cDNA
- cutting desired fragments out by using restriction endonucleases from the DNA
- using a gene machine
Explain how reverse transcriptase can be used to produce gene fragments
- the enzyme catalyses the production of DNA from RNA
- most cells only contain 2 copies of each gene making it difficult to obtain the DNA fragment containing the target gene
- mRNA is complementary to the gene and it is easier to obtain as there is loads of it
What are the advantages of using reverse transcriptase to produce DNA fragments
- uses mRNA from a cell, actively transcribing the gene - good as there is lots of mRNA
- mRNA doesn’t contain introns
What are the disadvantages of using reverse transcriptase to produce DNA fragments
- lots of steps involved so it takes more time and is technically more difficult
Explain how restriction endonucleases can be used to produced DNA fragments
- bacteria have special enzymes these are restriction endonucleases these are used as a defensive method to cut up DNA that may have been inserted by viruses
- the active site is complementary in shape to a range of different DNA sequences called recognition sites each of these are cut at specific places in DNA
What is a palindromic DNA sequence?
a sequence of antiparallel base pairs
What does restriction endonuclease do to the recognition sites?
restriction endonuclease recognises the site and cuts the DNA
the type of cut depends on the sequence of these sites
What are the two types of cut
- even cut
- uneven cut
What is an even cut?
a cut between 2 opposite base pairs resulting in blunt ends
What is an uneven cut?
a cut leaving sticky ends
What happens if the recognition sites are present either side of the target DNA?
- DNA is able to be incubated with a specific restriction endonuclease allowing the target DNA to be separated from the rest of the DNA this is done through hydrolysis reactions
Explain what the blunt and sticky end are used for?
they are able to be used to anneal (bind) the fragment to another piece of DNA that is complementary
What are the advantages of using restriction endonuclease to produce DNA fragments?
- sticky ends make it easier to insert the DNA into another organism to make recombinant DNA
What are the disadvantages of using restriction endonuclease to produce DNA fragments?
- still contains introns
Explain how the gene machine can be used to produce DNA fragments?
-bioinformatics - desired nucleotide sequence is fed into the computer
- synthesis of oligonucleotides
- assembly of gene - oligonucleotides are overlapped then joined together and made double-stranded using the PCR
- the gene is the inserted into a bacterial plasmid
- genes are sequenced and those with errors are rejected
- gene is usually delivered and incorporated into the plasmid
What are the advantages of using the gene machine to produce DNA fragments?
- ability to design exact fragments you want, intron free and can add sticky ends
- its a very quick and accurate process
What are the disadvantages of using the gene machine to produce DNA fragments?
- need to know the sequence of amino acids however there is a large database so this isnt a big problem
How can marker genes be used to identify whether a gene has been taken up by bacterial cells
- using a second separate gene on the plasmid
this gene may be identifiable for these reasons: - may be resistant to an antibiotic
- may make a fluorescent protein
- may produce an enzyme
Describe the process of gene transfer and cloning
- isolation - production of DNA fragments that have the required gene using reverse transcriptase or restriction endonucleases
- insertion - insertion of DNA fragment into a vector e.g. a plasmid using DNA ligase
- transformation - introduction of DNA fragment into suitable host cell
- identification - identification of host cells that have taken up the DNA using gene markers
- growth/cloning - culturing of host cells containing the DNA to produce the protein on a large scale
How can isolated DNA fragments be placed in plasmids
- plasmid and gene are cut with the same restriction enzyme to create complementary ends (sticky ends) this means that they inserted DNA and vector are complementary and can be joined
- the fragments are incubated with the plasmids. if a plasmid takes up the insert, base pairing takes place between the complementary ends which are then sealed with the use of DNA ligase which forms phosphodiester bonds
- a recombinant DNA molecule is created
