Restriction Endonucleases and Recombinant DNA Technology. Flashcards
Define a bacteriophage?
Viruses that affect bacteria.
Define a clone?
A group of cells that contain the exact same recombinant DNA sequence.
Define a cloning vector?
A DNA molecule that is capable of replicating inside a host cell.
Define competent cells?
Cells that are capable of having DNA added to their genome.
Define contigs?
DNA strands that have been cleaved to produce overlapping sequences.
I.e. they have not been cleaved at all of their restriction sites.
Define COS sites?
These are cohesion sites and they are another name for sticky ends.
Define an endonuclease?
Enzymes that cut phosphodiester bonds in the middle of a DNA molecule.
Define an exonuclease?
Enzymes that cut phosphodiester bonds at the ends of a DNA molecule.
Define a nuclease?
An enzyme that cleaves phosphodiester bonds in a nucleotide chain.
Define an oligonucleotide?
A polynucleotide whose molecules contain a small number of molecules.
Define phage 𝞬?
A particular bacteriophage.
Define phage induction?
The process that bacteriophages use to integrate their genome into a bacterial cells genome.
Define DNA recombinant technology?
When DNA sequences from many sources are bought together synthetically to create a new sequence of DNA that is not found naturally.
What molecules does DNA recombinant technology heavily rely on?
The use of recombinant DNA molecules.
Define restriction endonuclease’s?
Enzymes that recognise specific base pair sequences and cleave the DNA at that sequence.
Define a restriction site?
The site on DNA where endonuclease enzymes cleave the 2 strands.
What sequences of DNA usually make up restriction sites?
Short palindromic sequences.
Define the process of screening?
The process of searching for a specific DNA sequence in a DNA library.
Define a DNA vector?
A piece of DNA that is capable of independent growth.
DNA vectors are often what kinds of DNA?
Bacterial plasmids or viral phages.
Cloning the ability to manipulate what?
DNA that is then capable of reproducing itself.
How are bacterial plasmids used for recombinant processes?
By inserting plasmids into bacteria, this gets the genetic information in the plasmids translated into proteins.
E.g. insulin manufacture.
A plasmids self replicating?
Yes, they will divide every time binary fission occurs.
Can plasmids be intergrated into a bacterial genome?
Yes.
How do bacteriophages infect bacteria cells?
By injecting their viral genome into the cell.
How does the bacterial cell make viral proteins?
Viral DNA codes for the host cell machinery to make viral proteins that can be assembled into more viruses.
What shape is the DNA that is injected into bacteria by bacteriophages?
It is linear and contains a 5 prime and a 3 prime overhang at each end.
What are the 2 overhangs on a linear strand of viral DNA used for after it has been injected into a bacterial cell?
They are complimentary and form bonds to form a piece of DNA that is circular in shape.
How does the circular viral DNA incorporate itself into the viral genome?
It bimnds one of its own sequences of DNA to a complimentary sequence on the bacterial DNA.
What is the complimentary sequence between the viral DNA and the bacterial DNA known as?
The ATT site.
What happens once the ATT site on viral DNA has bound to the bacterial DNA?
The ATT site opens up and allows the viral DNA to become integrated to the bacterial genome.
What did scientists use to study bacteriophage’s?
They took an E.coli cell and infected it with a lambda phage.
The lambda phage replicated and eventually lysed the cell, releasing a load of baby viruses.
What happened when scientists were studying bacteriophage’s after the lambda phage had lysed the bacterial cell?
The baby viruses were used to infect K-12 cells.
What were the baby viruses called after the lambda phage had lysed the bacterial cell when scientists were studying bacteriophage’s?
Lambda-C viruses.
What happened during the study of bacteriophage’s after the lamda-C viruses had been used to infect K-12 cells?
The viral process only occurred in very rare cases, meaning that the K-12 cells were mainly unharmed.
Why did the viral process not occur in the K-12 cells during the study of bacteriophage’s?
The K-12 cells could recognise and destroy foreign DNA.
How could the K-12 cells recognise foreign DNA during the study of bacteriophage’s?
Their DNA is methylated, allowing them to differentiate between their own DNA and foreign unmethylated DNA.
How did lambda-C viruses infect some of the K-12 cells during the study of bacteriophage’s?
They modified their own DNA by methylating it.
This meant that the K-12 cell could no longer tell the difference between its own DNA and the phage DNA.
What enzymes do K-12 bacteria contain that helps them to distinguish between their own DNA and foreign DNA?
Endonuclease’s and methylase’s.
A methylase will methylate restriction sites.
The endonuclease will recognise these restriction sites and will cleave them if they are not methylated.
When does the methyl-transferase in a K-12 bacterial cell methylate bacteria?
After a restriction enzyme called ECO-R1 has cleaved the bacterial DNA at a specific site.
What are the sites in K-12 bacterial DNA that are cleaved by the restriction enzyme ECO-R1 so they can be methylated?
Palindromic sequences.
These sequences read the same on each strand.
What kind of methylase will methylate the palindromic sequences on K-12 bacteria?
An ECO-R1 methylase.
What is the enzyme that will re-attach the 2 bacterial strands after methylation has occurred in K-12 bacteria?
ECO-R1.
Why will palindromic sequences re-attach easily?
Because they form sticky ends.
How does the endonuclease in a K-12 bacterial cell protect the bacteria cell against viral invasion?
It scans the DNA within the genome and cleaves any un-methylated sequences.
What kind of viral DNA will not be recognised by the endonuclease in the K-12 bacterial cell?
When a virus modifies its own DNA to give the same methylation pattern that is on the host DNA.
The process that bacteria use to protect their DNA through methylation is known as what?
As bacterial restriction and modification.
Which scientists discovered bacterial restriction and modification?
Meselson and Yuan.
How did Meselson and Yuan discover bacterial restriction and modification?
They purified a bacterial enzyme that cleaved lambda-C phage DNA into reproducible pieces.
What did Meselson and Yuan name the bacterial enzyme that cleaved lambda-C phage DNA into reproducible pieces
A restriction endonuclease.
How does a restriction endonculease in bacteria recongnise bacterial DNA?
Via methylation, which bacteria do in a species specific manner.
How do the daughter strands of bacterial DNA become methylated after cell division?
After division, the parent strand remains methylated and the daughter strand will quickly become methylated.
What kind of molecules are restriction endonuclease enzyme?
Dimers.
What are restriction endonuclease enzymes often used as tools for?
For cloning and other recombinant techniques.
What are the 3 major types of restriction endonuclease?
Type 1.
Type 2.
Type 3.
How common are type-1 endonculease’s?
Less common than the type 2 endonuclease and more common than the type 3.
How do type-1 endonculease’s cut DNA strands?
They cut both DNA strands at a random location which is far from the recognition site.
Are type-1 endonculease’s often used in recombinant technology?
No.
What are the most common types of endonculease?
Type-2.
What are the most frequently used endonuclease’s in recombinant technology?
Type-2.
How do type-2 endonculease’s cut DNA strands?
They cut both DNA strands at specific locations which are usually palindromic and around 4-8 base pairs long.
Will type-2 endonculease’s create sticky ends when they cut the DNA strands?
Yes.
What are the rarest types of endonculease?
Type-3.
Are type-3 endonculease’s often used in recombinant technology?
No.
How do type-3 endonculease’s cut DNA strands?
They cut a single DNA strand that is around 25 base pairs downstream of the recognition site.
Why are type-1 and type-3 endonuclease’s rarely used in recombinant technology?
As they are said to be promiscuous, meaning that they cause random cleavage patterns.
Which endonulcease’s are said to be the basic tools of cloning?
The type 2’, as they will only cleave at specific sites.
How is a restriction endonuclease named?
The first 3 letters are named after the organism that they were found in.
The 4th letter represents the strain of endonuclease.
The letters are followed by a number to indicate the order of discovery of endonuclease’s in that organism.
E.g. ECO-R1 was found in E.coli and it was the first restriction endonuclease to be found in E.coli.
What do the recognition sites for type-2 endonculease’s tend to be?
Around 6 base pairs and they are palindromic.
How will a palindromic sequence read on each strand?
They will read exactly the same in the 5 to 3 direction on both strands.
What was does an endonuclease read a DNA strand?
In the 5 to 3 direction.
What is the most common feature on a DNA strand that has been spliced by a type 2 endonuclease?
To create sticky ends, where there is an overhang on each strand.
Why do type II endonuclease’s often create sticky ends on each strand?
Because the overhangs are complimentary menaing that the 2 strands will get back together easily.
Describe what happens when blunt ends are formed by a restrioction endonuclease?
Blunt ends are created when neither strand has an overhang and these strands are very difficult to re-join.
How do researchers predict the probability of getting the right nucleotides for a recognition sequence that reads 5-CCGG-3?
The probability would be 1/4 that the 1st nucleotide is a C.
This multiplied by 1/4 that the 2nd nucleotide is a C.
This is multiplied by 1/4 that the 3rd nucleotide is a G.
This is multiplied by 1/4 that the 4th nucleotide is a G.
Probability of a 5-CCGG-3 match occurring is 1/4 * 1/4 * 1/4 * 1/4 = 1/256.
This means that a recognition site of 4 base pairs is likely to occur every 256 base pairs.
How does the amount of base pairs in a recognition site affect the number of nucleotides in between each site?
The more nucleotides in the recognition site means more base pairs between each site.
Which enzyme joins the nucleotides together after they have been cut by a restriction endonculease?
DNA ligase catalyses the formation of a phosphodiester bond so that nucleotides can be joined together.
How does DNA ligase form a phosphodiester bond between the 2 nucleotides?
By joining the 5-prime phosphate on one nucleotide with a 3-prime hydroxyl group on another.
