chapter 21 - recombinant DNA technology Flashcards
what is recombinant DNA
DNA of two different organisms that has been combined. isolate genes. clone genes. and transfer them into micro organisms.
what is an organism formed from recombinant DNA called.
known as a transgenic / genetically modified organism (GMO)
why is possible that DNA of one organism is accepted by a different species but also functions normally when it is transferred
because the genetic code is the same in all organisms. it is universal. making the proteins is also universal as the mechanisms of transcription and translation are essentially the same in all organisms. therefore transferred DNA can be transcribed and translated within the cells of the recipient (transgenic organism)
describe the process of making a protein using the DNA technology of gene transfer and cloning
isolation- of the DNA fragments, that have the gene for the desired protein.
insertion- of the DNA fragment into a vector
transformation- transfer of DNA into a suitable host cell
identification- of the host cells that have successfully taken up the gene by the use of gene markers
growth/cloning- of the population of host cells
What is the meaning of sequencing a genome
Working out all the DNA base sequences for all of the DNA in the cell
How long did the human genome take to complete
13 years
Give an example of a method that is used to sequence a genome
The sanger method
We do not specifically need to know the methods for AQA, however the methods are continuously being improved and updated. At this current moment they are mostly automated.
Why is it easier to work out the proteome for prokaryotes
because prokaryotes contain no introns. Therefore the genome is used directly to work out the proteome. This is useful to create vaccines as the antigens can be worked out directly.
Why is it harder to translate the proteome in eukaryotes.
Eukaryotes have Intron’s and regulatory genes in DNA. Therefore the genome cannot be easily used to translate the proteome.
Why is recombinant DNA technology used
It is used to improve industrial processes and medical treatment
What are three methods to creating fragments of DNA during isolation
- conversion of mRNA to cDNA using reverse transcriptase.
- Using restriction endonucleases to splice out desired genes from DNA.
- Using a gene machine to create desired genes [based on protein structure]
What are retroviruses
They are a group of viruses [HIV]
Describe the process of using reverse transcriptase to isolate a gene
- A cell that naturally produces the proteins of interest is selected
- beta cells in the islets of Langerhans in the human pancreas are specialized to produce insulin, they make a lot of mRNA that codes for insulin.
- Messenger RNA code for insulin
- MRNA acts as a template on which a single stranded complementary copy of DNA is formed.
- reverse transcriptase joins complementary DNA nucleotides and cDNA is formed [single-stranded]
- cDNA is isolated via the hydrolysis of mRNA with an enzyme
- double stranded DNA is formed on the template of the sea DNA using DNA polymerase.
- A copy of the human insulin gene is made
What is the advantage of cDNA
It is intron free is it is based on the mRNA template
What does cDNA stand for
Complementary DNA
What are restriction endonucleases
They are enzymes that cut up DNA
Where are restriction endonucleases found
They naturally occur in bacteria as a defense mechanism
What is the site called by restriction endonucleases cut
The recognition sites/sequence
What type of ends are left when restriction endonucleases cut at the same location on DNA
Blunt ends
What type of ends are left one restriction endonucleases cut in a staggered fashion
Sticky ends
what are exposed staggered ends called
Palindromic/palindromic sequence
What is the importance of sticky ends
They have the ability to join to DNA with complementary base pairs
What does a palindromic sequence mean
It’s reads the same forwards as it does backwards
Which type of restriction endonuclease is more useful [one that produces a palindromic sequence/sticky ends or one that produces blunt ends]
One that produces a palindromic sequence/sticky ends.
When this section of DNA is placed into a new organism it’s can more easily combine with the original DNA of that organism.
It makes it easier to join the DNA with the next sample
Describe the process of creating a DNA fragments via a gene machine
Scientist would examine the protein of interest and determine the primary structure [amino acid sequence] from this they were determined the mRNA sequence and then the DNA sequence that follows.
The DNA sequence is entered into a computer and the sequence is checked for biosafety and bio security to ensure it meets international standards as well as various ethical requirements.
The computer designs a series of small, overlapping single strands of nucleotides called oligonucleotides. Which can be assembled into the desired genes.

next, in an automated process, each of the oligonucleotides is assembled by adding one nucleotide at a time in the required sequence.
The oligonucleotides are then join together to make the desired gene. The stream does not contain introns or non-coding DNA.
The chain is replicated using the polymerase chain reaction [PCR]
The polymerase chain reaction also constructs the complementary strand of nucleotides to make the required double strand of gene. Using sticky ends the gene can be inserted into a bacterial plasmid. This acts as a vector for the gene allowing it to be stored, cloned or transferred to another organism in the future the jeans are checked using standard sequencing techniques and those with errors are rejected.
What is the advantage of producing a DNA fragments by the gene machine
That any sequence of nucleotides can be produced in a very short time [as little as 10 days] and with great accuracy
a further advantage is that these artificial genes are also free of introns and other non-coding data that’s all they can be transcribed and translated by prokaryotic cells.
What are the advantages of using reverse transcriptase
The mRNA from a cell has been actively transcribed, the availability of mRNA is used to make cDNA.
What are the disadvantages of using reverse transcriptase
It is more time-consuming therefore it is more technical difficult
What are the advantages of using restriction endonucleases
Sticky ends on a DNA fragments make it easier to insert and make it easier to make recombinant DNA
What are the disadvantages of using restriction endonucleases
it still contains introns
What are The advantages of using a gene machine
You can design the exact DNA fragments required with sticky ends and preferential codons.
What is the disadvantage of using a gene machine
You need to know the amino acid sequence/primary structure/sequence
What is in vivo
It is transferring the fragments to a host cell using a vector
What is in vitro
Using the polymerase chain reaction [PCR]
Describe the process of using sticky ends to combine DNA from different sources
The DNA double helix is cut at the recognition site with restriction endonuclease. DNA from another source cut with the same restriction endonuclease Joins with the other DNA fragment via DNA ligase which joins the two sections forming recombinant DNA.
What is the function of DNA ligase
It is used to bind the phosphate sugar framework of the two sections of DNA to form recombinant DNA. Sticky ends have considerable importance because, provided the same restriction endonucleases used. We can combine the DNA of one organism with that of any other organism.
how do we prepare the DNA fragments for incision (in vivo)
we add a promoter region - To the start of the DNA fragment, this is a sequence of DNA which is the binding site for DNA polymerase to enable transcription to occur.
We also add a terminator region - To the end of a gene/fragment. Its causes RNA polymerase to detach and stop transcription. So only one gene at a time can be copied into mRNA.
What is a vector
Something that can carry the isolated DNA fragment into the host cell
What are the most common types of vector
Plasmids [circular DNA, separate from the main bacterial genome which only contains a few genes.]
Describe the process of incision of a DNA fragment into a factor
The plasmid is cut open using the same restriction endonuclease
therefore the same sticky ends are formed
DNA fragments sticky ends/expose nucleotides are complementary to the sticky ends on the plasmid
Therefore the DNA fragment is now attached to the plasmid
DNA ligase sticks plasmid and the DNA fragment together [Annealing them]
DNA ligase catalyzes a condensation reaction to form phosphodiester bonds between nucleotides.
Describe the overall process of in vivo Cloning
create DNA fragments of the gene of interest
Insert DNA fragments into a vector
Transform the host cell with a vector
Identify the transformed cells
Go to the host cell, clone/make copies of the gene.
What does transformation mean
It means getting a plasmid inside the bacteria/host cell. the gene will be expressed on the Protein will be made.
How do we insert the plasmid into the host cell
to do this the cell membrane of the host cell must be more permeable
To do this we mix The host cells with calcium ions and we also heat shot this mixture (a so an increase in temperature)
This enables a vector to enter the host cells cytoplasm.