Recombinant Technology Flashcards
What are ‘restriction enzymes’?
Enzymes which reliantly cut DNA into more manageable sizes, by cutting at precise recognition sequences (palindromic sequences)
Where were most restriction enzymes discovered?
In bacteria
What is an example of a restriction enzyme?
Xbal
How long are the recognition sequences of restriction enzymes?
6 nucleotides lons
How do restriction enzymes usually act?
As dimers
What 2 ways can DIFFERENT restriction enzymes cut DNA?
1) Sticky-ends: cut through the DNA backbone asymmetrically, leaving overhangs
2) Blunt: Cut symmetrically, flush ends
How can restriction fragments be separated from each other?
What is the process of this?
Gel electrophoresis:
1) Place sample in the well in the top of the agarose gel (in buffer solution
2) Apply a current across the gel
3) The negatively charged DNA is attracted to the positive anode
4) As the fragments move, they become separated by size (smaller travel faster as they can pass through smaller holes)
5) Stain the gel using florsecence (eithidium brominde)
6) End up with many bands on one lane
- Can run many samples on one gel
7) Cut the DNA out of the gel
What gel is used in gel electrophoresis to separate restriction fragments?
Agarose gel
Using gel electrophoresis, how can the length of fragments be worked out?
Run known sized fragments on the gel and compare with the bands of the unknown fragments
What is ligase?
An enzyme which joins 2 DNA fragments together to form recombinant DNA, using ATP
What is recombinant DNA?
DNA which doesn’t normally exist in the nature - formed by laboratory methods, which bring together DNA from multiple sources
How does ligation work better?
If the fragments of DNA have the same overhang (cut by the same restriction enzyme) as they can hybridise with each other and form H bonds
What is another way to explain ‘sticky ends’?
Cohesive termini
What are plasmids?
Small, circular, extra-chromosomal DNA that occur naturally in bacteria
They have a single origin of replication
How many copies of the plasmid is made in bacteria?
50
What does cloning DNA involve?
Ligation of DNA fragments into a plasmid vector
What do plasmid vectors usually carry?
Antibiotic resistance, which is shared between strains of bacteria
How are plasmids used to grow a large amount of small DNA fragments?
Why is this needed?
1) Engineer in many enzyme cut sites (to form a multiple cloning site)
2) Digest the plasmid vector with the required restriction enzyme
3) Insert in the DNA of interest - create a recombinant plasmid
4) Insert the plasmid back into the bacteria - each cell will make about 50 copies of the plasmid
5) Single colony lifted off the plate to start a liquid culture
6) Plasmid purified from the bacteria and stored or analysed
Needed in order to make many copies of a DNA fragment - to be able to study
What is a ‘multiple cloning site’?
DNA region within a plasmid which contains multiple UNIQUE restriction enzyme cut sites - to allow foreign DNA to be inserted
How much DNA can plasmids hold?
What happens if too much is added?
<30 kilobases
If too much - bacteria get sick, as they don’t have enough energy to produce that much DNA
What can be used to add in more DNA to clone it? (2 things)
How much DNA can they hold?
What is the problem with these?
1) Bacterial Artificial Chromosome (<300 kilobases)
2) Yeast Artificial Chromosome (<3 megabases)
Larger chromosomes are more fragile and harder to work with
What is transformation?
Inserting the plasmid (containing the DNA of interest) into the bacteria
How is bacteria transformed?
By creating temporary holes in the cell membrane of the bacteria and mixing it with the plasmid containing the DNA
How are holes made in the bacterial membrane, in order for the plasmid to be incorporated into it?
Electroporation - electric charge makes holes
OR by
Chemical treatment
How do you select for bacteria that have taken up the plasmid?
By treating the bacteria on antibiotic plates - as the plasmid contains the gene for antibiotic resistant gene
Bacteria without the plasmid will die
What is cloning?
Making multiple, identical copies of a single DNA fragment (plasmid)
Where do we get DNA to start with to use in the lab? (2 places)
1) Library of genomic clones
2) Library of cDNA clones from mRNA
What does a genomic library contain?
Fragments of genomic DNA - ALL the genes in the genome
How is a genomic library formed?
By digesting DNA from tissue (using restriction enzymes) and cloning fragments in plasmids
If this is done enough times - get the entire genome
What are the advantages of a genomic library?
Contains all the REGULATORY sequence (introns)
So can study transcriptional regulation
What does a cDNA library contain?
Genes which are expressed in different cells - represents all the mRNA of a given cell type
What are the advantages of a cDNA library?
Can compare the genes expressed in normal tissue and diseased tissue
Can identify genes involved in disease
How is a cDNA library formed?
1) Extract mRNA from a cell
2) Using reverse transcriptase make cDNA
3) Ligate cDNA into a plasmid vector and transform into bacteria
4) Grow the bacteria culture and purify out the DNA clone
Where is reverse transcriptase found?
In retroviruses:
RNA into ssDNA
ssDNA into dsDNA
What is cDNA?
DNA made from mRNA - doesn’t contain any introns/promoters/regulatory elements (only the exons)
What is the ‘transcriptome’?
All of the mRNA in a cell (all of the genes expressed)
Different in different cells
When making a cDNA or genomic library, what is it important to ensure?
That there is only:
One insert in each plasmid
One plasmid in each bacteria
One bacteria starting each colony
So that one bacteria is representing ONE mRNA
What types of genes will be expressed by different colonies when making a cDNA library?
1) Housekeeping genes
2) Tissue specific genes
Why when making cDNA libraries, do you end up with many colonies of housekeeping genes?
These genes are more common and are highly transcribed, so there is many copies of the mRNA in the cell
What are ESTs?
How are they used?
Expressed Sequence Tags:
- Small pieces of DNA generated by sequencing randomly selected clones from a cDNA library
- Used to identify what DNA is present in the colony and particular genes of interest
What is a genomic library useful for?
Sequencing genomes
When making genomic libraries, what is it possible to get at the end?
An individual bacteria clone which represents the entire genome
What is the process of dideoxy terminator sequencing?
1) Denature (heat to 100 degrees) the template (DNA which want to identify) to make it single stranded
2) Allow the DNA to cool with a primer
3) DNA synthesis reaction with DNA polymerase, dNTPs and a ddNTP of a certain base (A,G,T or C) - do this in 4 different tubes
4) Different strands will terminate at different positions
5) Run ALL the samples of the gel - forms separate the bands, depending upon the size of the fragment
6) Can see the full length of the DNA as the DNA fragment is COMPLIMENTARY to the ddNTP sequence
Describe the primer, added in dideoxy terminator sequencing
Short, single stranded DNA that can be easily synthesised
Designed to anneal against the edge of the vector
Extended by DNA polymerase
What are ddNTPs and how are they different to dNTPs?
dNTPs have -OH at the 3’ end - so that oxygen can join to the phosphate and the DNA can elongate
ddNTPs have -H at the 3’ end (no O to join to phosphate) - Cannot be elongated and the DNA polymerase falls off
- Termination of the DNA sequence
What is the process of AUTOMATED dideoxy terminator sequencing?
1) Label each ddNTP with a different fluorescent dye
2) Termination will be labelled in a colour corresponding to the nucleotide which terminated it
3) As pass through the gel - camera takes pictures of each band and measures its intensity (one position over time, not the whole gel)
4) Produces a graph (trace) showing colour intensity over time
What size DNA can automated dideoxy terminator sequencing analyse?
What can be used if the size of the DNA is above this?
<1000 (1kb)
If above 1kb, can use other methods:
1) Progressive sequencing
2) Shotgun sequencing
What is the process of progressive sequencing?
1) From a GENOMIC library, sequence the ends of the clones, using primers from the vector
2) Synthesise primes on each end - reading in
3) Design a primer based upon the new sequence - another round of sequencing is performed
4) Keep doing this until the primers meet in the middle - eventually get sequence of the whole fragment
What is the full process of determining the function o DNA in the genome?
1) Source DNA or mRNA
2) Insert into plasmids and clone
3) Extract a sample of interest (can do this be sequencing the end - ESTs
4) Make a library (genomic or cDNA)
5) Sequence the DNA (dideoxy or progressive/shotgun for larger pieces of DNA)
Why in progressive sequencing must primers of the VECTOR be used?
The vector has a known sequence
What is needed in order to sequence a genome?
Many BAC clones (bacterial artificial chromosome), which can be localised to particular regions of the genome - chosen for sequencing
What are the advantages of shotgun sequencing?
Requires no thought - automated primer design
What are the disadvantages of shotgun sequencing?
Need to sequence more than 6X the size of the genome to get large contigs
Always be gaps
What is the process of shotgun sequencing?
1) Make genomic plasmid libary
2) Sequence just the ENDS of the inserts (using a primer of the vector)
3) Use the same primer for the ENTIRE library (matches the vector - same throughout library)
4) End up with many random sequences
5) Short random sequences are assembled by a computer into a contig (continuous sequence) -based on OVERLAP of the small sequences
How are shotgun sequencing and progressive sequencing used?
In parallel
