DNA Molecular Techniques

Outcome 1

DNA Cloning

The isolation of a specific DNA fragment (e.g. a gene) from cellular DNA. Or the amplification of the cloned DNA fragment

Clone

The DNA fragment produced by DNA cloning

Average Gene length

10,000-15,000 base pairs

DNA fragmentation

Breaking down genomic DNA into smaller pieces which are easier to manipulate

Physical Shearing

Methods such as sonication, pipetting, or mixing which physically break up the DNA into random length smaller pieces

Restriction enzyme digestion

The double stranded DNA (dsDNA) is cut at specific short sequences by restriction enzymes to provide a number of smaller fragments

Physical Shearing involves

Exposing DNA to shearing forces which progressively break the DNA down into smaller fragments. The application of force can be controlled, for example by pipetting the DNA sample for a certain number of times, by sonicating for a certain time period or by mixing with a vortexer for a certain time at a set speed.

Physical Shearing methods generally produce

DNA fragments of various lengths with blunt ends

Restriction Enzymes

A family of site-specific nucleases, they cleave/hydrolyse phosphodiester bonds in the DNA backbone at specific DNA sequences that tend to be Palindromic and 6-8 bp in length. 

Recognition Sites

The specific sequences that recognition enzymes cleave/hydrolyse phosphodiester bonds in the DNA backbone. Tend to be Palindromic and 6-8bp in length 

Unmethylated DNA

DNA that has not been covalently modified to contain a methyl group, usually to an adenine or cytosine base. Restriction enzymes can’t cleave methylated DNA.

Methylases

Enzymes that add methyl groups to DNA bases, protecting them from hydrolytic degradation by restriction enzymes.

“Endonucleases”

Classification of Restriction Enzymes because the phosphodiester bond cleavage occurs within a DNA chain and not at the chain terminus. Restriction endonucleases are therefore active on circular and linear DNA.

Palindromic Sequences

Those in which the base pair sequence of both strands in the recognition site are identical when read in the same direction (e.g. 5’ to 3’). Because there are two recognition sequences in the palindromic site of double stranded DNA, a restriction enzyme will cleave both strands.

Hpal

a restriction enzyme which cleaves a six-nucleotide recognition sequence in dsDNA. Hpal —→ Haemophilus PArainfluenzae

¼ * ¼ * ¼ ¼ = 1/256

Frequency of a four base sequence appearing

4kb or 4096 bp

The length of DNA fragments a long DNA fragment would be cut into on average by a restriction enzyme that recognises a 6 base recognition sequence. these fragments are known as restriction fragments.

Blunt Ends

Occur when a restriction enzyme cuts through the sugar phosphate backbones at direct opposite sides of both DNA strands.

Sticky Ends

Occur when restriction enzymes such as EcoRI cuts both DNA backbones at different points (not directly through the centre of the Palindromic site). The single stranded extensions produced are called “sticky ends” or “overhangs” If the overhang extends towards the 5’ end its called a “5’ overhang”

Overhangs/Sticky Ends Can

Hydrogen bond together to reform dsDNA. However hydrogen bonds can break. The original cut (i.e. the P-O covalent bond) must be repaired to complete the sugar-phosphate backbone. This can be carried out by an enzyme called DNA ligase.

DNA Ligase

Catalyses the formation of a phosphodiester bond between two dsDNA molecules to join them together provided that one has an exposed phosphate group at the 5’ end and the other has a free OH group at the 3’ end. DNA Ligase can repair (or ligate) breaks in dsDNA molecules and therefore performs the opposite function to a restriction enzyme.

DNA Ligase can be used to

Covalently bond the sticky ends of DNA together after restriction digestion. It can covalently bond any pieces of DNA provided that they have complimentary sticky ends or blunt ends. So it can be used to circularize a plasmid after it has been linearized by restriction digestion.

Sticky ends can be converted to Blunt Ends

Blunt Ends are typically produced during physical shearing processes but some fragments may have random overhangs. Under certain circumstances, it is useful to convert sticky ends into blunt ends so that all DNA fragments have uniform ends.

The Klenow fragment of E. coli DNA ploymerase can

Produce Blunt Ends from overhangs. This enzyme catalyses 5’-to-3’ polymerization which means that it can make a 5’ overhang blunt by ‘filling in’ receded 3’ ends of DNA fragments.

The Klenow fragment requires

A DNA template and a mixture of deoxynucleotides: dCTP, dGTP, dCTP and dTTP, Mg²+ and a free 3’-OH terminus on the DNA strand which is being synthesised.

Like DNA Polymerase, the Klenow Fragment

Catalyses the formation of a phosphodiester bonds between the 3’ hydroxyl group of the terminal nucleotide and the 5’ phosphate of the dNTP (this is known as 5’—>3’ polymerase activity).

Restriction Maps

A description of the restriction sites within a piece of DNA. It is often the first step used to characterise unknown DNA e.g. a gene of interest

Bacterial Plasmids

Typically circular dsDNA molecules separate from the chromosomal DNA and capable of autonomous replication

Plasmid Replication

Independent of Chromosomal Replication due to Origin of Replication (Ori) sequence. Plasmids also contain restriction enzyme recognition sequences which can be cut with specific restriction enzymes to allow for the insertion of foreign/exogenous DNA.

amp^r gene

Codes for the Beta lactamase enzyme which hydrolyses Beta lactam antibiotics such as ampicillin.

Antibiotic Resistance Genes are

Engineered to Plasmids, where they act as selectable markers i.e. they allow for the selection of bacteria expressing a plasmid

Vector

The vehicle the cloned DNA fragments are routinely stably stored in and transported in during Cloning such as a plasmid

Agarose Gel Electrophoresis

Agarose a polysaccharide in solid gel form when in aqueous solution is placed into an electrophoresis chamber and a conductive buffer is added to cover the gel. The DNA sample(s) being analysed/separated must be prepared by adding tracking dyes to allow visualisation of how far the DNA has migrated down the gel during electrophoresis. Sucrose or glycerol are added to the gel to make the sample dense, preventing diffusion of the DNA back.

During Electrophoresis

DNA fragments of different sizes will be separated on the basis of size and charge. DNA is anionic and therefore all DNA will migrate toward the anode (+). 

The Agarose gel behaves like a

Sieve during the experiment, separating large and small DNA fragments from each other. Large DNA fragments (such as chromosomal DNA) will be impeded by the agarose network whereas smaller DNA fragments (e.g. human insulin gene) can more readily migrate through the agarose network further toward the anode.

The Tracking Dye (Dark Blue usually)

Allows the progress of the electrophoresis to be followed. After electrophoresis, the agarose gel is stained with intercalating dye like ethidium bromide or with coloured dyes which bind to the DNA. The DNA fragments will be observed as “bands” that are then visualised by illuminating the stained gel under UV (EB) or visible light.

The band of interest

Corresponds to the gene of interest (e.g. human insulin) can be cut from the gel using a scalpel for removal from the agarose and subsequent use i.e. litigation into a linearised vector.

Plasmid Self-Ligation

Major problem with ligation of sticky ends is that it is possible for sticky ends on the same linearised plasmid (or sufficiently long DNA fragments) to join together or ‘self ligate’. In this case the plasmid will reform without the Cloned DNA fragment. Both the recombinant plasmid containing the cloned DNA fragment and the Self-Ligated Plasmid would exist within the same sample. Only the recombinant plasmid would be of interest to scientists.

Preventing Plasmid Self-Ligation

Self-Ligation is dependent on the concentration of DNA. High Plasmid Concentrations favour Self-Ligation. An Insert: Plasmid ratio of 3:1 favours intermolecular Ligation and produces more Recombinant Plasmids.

Presence of Phosphate Groups on 5’ ends of DNA is required for Ligation

If Phosphate groups are removed from the plasmid termini then Plasmid Self-Ligation cannot occur. 

Alkaline Phosphatase 

An enzyme that removes phosphate groups (Dephosphorylates) the linearised plasmid at the 5’ ends of both DNA strands, which prevents Self-Ligation. Insert DNA still contains phosphate groups so it can still ligate to the vector. Only Alkaline Phosphatase-treated vectors that have been ligated to inserts can circularize and replicate.

The process of cloning is not complete after the generation of a Recombinant Plasmid

To facilitate the amplification of and characterisation of the cloned DNA Fragment, the Recombinant Plasmid is often introduced to Bacterial Cells. A process known as “Bacterial Transformation” is performed to transfer the Recombinant Plasmid to Bacterial Cells. A Transformation is one example of horizontal gene transfer, that involves the uptake of exogenous (Recombinant Plasmid) DNA to living Bacterial Cell.

DNA is a Hydrophilic Molecule

It cannot normally pass through the bacterial cell membrane. Cells that are capable of being transformed are called ‘Competent’. Transformation can occur naturally between certain bacterial strains but this is a rare occurrence because very few bacterial strains are naturally competent and those that aren’t must b made competent prior to induced transformation.

Chemical Transformation

Method of Transforming Bacteria. Cells are incubated in ice cold solution containing calcium chloride, the recombinant plasmid DNA is added. Cells are then subjected to brief heat shock (e.g. at 42^oC for 30-45s) in the presence of the recombinant plasmid DNA. 

Heat Shock

Generates Temporary pores in the cell envelope allowing the recombinant Plasmid DNA to move into the cells.

The presence of divalent cations such as calcium in Chemical transformation is necessary 

To neutralise the negatively charged phosphates on Recombinant Plasmid DNA and the negatively charged phosphate/carboxylate groups on the cell wall and membrane, reducing charge-charge repulsion between these negatively charged groups, allowing the plasmid to move close in space to the bacterial envelope

Following Heat Shock and return to the ice bath

Pre-warmed recovery broth is added to the cell slurry and the now culture is incubated at 37^oC for 30-45 minutes before plating out on solid growth medium. only transformed cells will carry the selectable marker that will allow them to survive on the growth medium inoculated with e.g. ampicillin. This is an example of positive selection

Electroporation

Exposing cells to a short electric shock which appears to briefly open holes or pores in the cell wall/membrane. Allows for DNA to enter the cells and they can then be plated out for selection.

Transformation Efficiency 

The number of colony forming units (CFU) produced per microgram of recombinant DNA used to transform the bacteria.

Effect of plasmid size on Transformation efficiency 

As the size of plasmid increases, efficiency generally decreases since it becomes more difficult for the DNA to enter the cells. Cloning large pieces of DNA can be more difficult since they increase the size of the plasmid after insertion.

Effect of bacterial cell damage on Transformation efficiency 

Bacterial cells can become very fragile while being made competent. If they are damaged then the transformation efficiency will decrease

Method of Chemical Transformation effect on Transformation Efficiency

Electroporation is more efficient than Chemical Transformation

Incubation Time and Temperature effects on Transformation Efficiency

If Incubation time is too long the DNA may be degraded. If the temperature is too high, the cells may die.

Gene Library

A collection of cloned DNA fragments in which the DNA fragments are stored in a population of identical vectors, each containing a different insert of DNA. Mechanism for the storage of clones/DNA from a particular source.

Genomic Library

Contains cloned DNA fragments that together represent the genome of an organism of interest.

cDNA Library

Contains complementary DNA inserts synthesized from mRNA molecules from a cell or organism. These represent the expressed genes from the genome.

DNA Library Fragmenting Method should be

Restriction enzymes because of their specificity 

Fractionating of Genomic DNA Fragments

After fragmentation the Genomic DNA Fragments are often fractionated (separated on the basis of size) to select correctly sized cloned DNA fragments

Following Fractionation

DNA fragments are ready to be inserted into cloning vectors

Cloning Vectors

A small piece of DNA that can be stably maintained and independently replicated within an organism.

As Genomic DNA has been cut with a restriction enzyme

The cloning vector is cut with the same enzyme to produce complementary sticky ends.

To create Eukaryotic libraries alternative vectors are generally used because

They can incorporate larger pieces of DNA insert and the Transformation Efficiency is greatly increased compared with plasmid transformation.

Representative Library

Represents the whole genome i.e. every sequence of DNA from the genome is represented by at least one clone

The number of  Recombinant Clones which must be produced to ensure full representation of the genome is dependent on

Average Insert size and Size of the Genome being screened

Clarke and Carbon Formula

N = ln(1-P)/ln(1-f)

Master Plate

Plate stored long term at -70 → -80^oC. Each Colony on the master plate carries a vector containing a different cloned DNA fragment. Using cells to carry the vector allows easy amplification and analysis.

DNA Hybridization

Possible to locate clones of a gene if the nucleotide sequence for part of the target gene is available. Single Stranded DNA Probe complementary to known short sequence of target gene