Lectures 4 + 5: Lecture Notes + Textbook
Clone: - Identical copy of something (ie exact copy of DNA)
- Needed for sequencing
- Bacteria reproduce by cloning themselves
- One example of synthetic cloning is PCR
The basic tools of gene exploration include
a) Restriction enzyme analysis
b) Blotting techniques (ie Southern blotting for DNA, Northern blotting for RNA)
c) DNA sequencing
d) PCR (polymerase chain reaction)
Restriction Enzymes
- Also known as restriction endonucleases
- Discovered by Arber and Smith
- Nathans pioneered their use in the late 1960s
- Recognize specific base sequences in double helical DNA, and cleave, at specific sites, both strands of a duplex containing the recognized sequences
- Extremely important – used in analyzing chromosome structure, sequencing DNA, isolating genes, and creating new DNA for cloning
- Restriction enzymes are naturally found in a wide range of prokaryotes
- They serve to cleave foreign DNA – the organism’s own DNA is not cleaved by its own restriction enzymes though because the sites recognized by the restriction enzymes are protected through methylation (only on own DNA)
- Many restriction enzymes recognize specific sequences of four to eight base pairs
- They act through hydrolyzing the phosphodiester bond in each strand
- Usually, rotational /bilateral symmetry – that is, the recognized site is palindromic (inverted repeat)
- For example, in the top picture to the right:
- Notice how the top strand and bottom one are inverses of each other
- The ◊ represents axis of symmetry (horizontal)
- The restriction enzyme will usually cut somewhere in the shaded area
- It always cuts the bond on the 3’ side of the axis of symmetry
- Thus, if this enzyme hydrolyzes the bond between the G and A, the
newly cut enzyme will look like:
- (It only hydrolyzes the 3’ region of each single strand)
- Notice the staggered DNA that is created – these are sticky ends
(more on that later)
- Restriction Enzymes usually recognize short base sequences
(short segments = higher probability of that sequence appearing multiple times = more clones
long segments = lower probability of that sequence appearing multiple times = less clones)
Southern Blotting
- Used to identify a specific sequence located within a DNA fragment
- First, a mixture of restriction fragments is separated by electrophoresis through agarose gel
- It is then denatured to form single stranded DNA, and transferred to a nitrocellulose sheet
- The positions of the DNA fragments are preserved on the nitrocellulose sheet
- These single stranded DNA are then exposed to a 32P labeled probe (radioactive probe)
- The probe hybridizes with restriction fragments having its complementary sequence
- Autoradiography displays position of this probe-fragment duplex
- RNA blot is called a Northern Blot, protein blots are called Western Blots
DNA Sequencing – the Sanger Dideoxy Method
- Developed by Sanger and co
- Works through controlled interruption of enzyme replication
- This procedure is performed by four reaction mixtures at the same time
- In all of these mixtures, DNA polymerase is used to make the complement of a particular sequence
- It is primed by a fragment that contains the complementary sequence to a known sequence
- What makes this method special is its use of specific “terminators” – each reaction contains a small amount of a 2’-3’ –dideoxy analog of one of the nucleotides (a different nucleotide for each reaction mixture – remember, there are 4 reaction mixtures, so 1 mixture will have the adenine form of the dideoxy analog, another will have the guanine form, and the other two will have thymine and cytosine)
- Normally, the polymerase adds a complementary base, then moves to the next nucleotide and does the same
- However, in addition to the regular nucleotides that the DNA polymerase usually add, they can also add these 2’-3’ –dideoxy analogs, as long as the base is the same. For example, if it needs to add an adenine to complement a thymine, it can also add a 2’-3’ – dideoxy analog of adenine (but it can not add a dideoxy version of guanine to complement thymine)
- However, if the dideoxy analog version of the nucleotide base is added instead of the pure nucleotide base, the polymerase is no longer able to add a nucleotide after it (this phenomenon is known as chain termination). The reason is that this dideoxy analog version of the nucleotide base does not have the OH group at the 3’ end, and thus, polymerase can no longer add bases
- The concentration of this dideoxy analog is low enough though that this “chain termination” will only take place occasionally (ie most of the times polymerase will add the regular nucleotide, but sometimes it will add this dideoxy version)
- Thus, different length fragments will be produced, but all of them will be terminated by a dideoxy analog base, and as such, these fragments of different length will correspond to the positions of its complement base on the regular DNA (sorry, I probably made this confusing – for example, the dideoxy analog dGTP (guanine base) would correspond to where a C would occur in the DNA sequence)
- Four sets (one for each base) of these chain terminated fragments then undergo electrophoresis, and the base sequence of the new DNA is read from the autoradiogram of the four lanes (remember, this is the new DNA – the sequence of the old DNA would be the complement to this new DNA)
- Fluorescent tags can also be used – it can either be attached to the priming fragment (a different color for each of the four reaction mixtures), or to the dideoxy analog itself (a different color for each of the four analogs)
- If added to the priming fragment, the four reactions are combined, then subjected to electrophoresis, and the separated bands of the DNA are detected by their fluorescence as they emerge from the gel
- If added to the dideoxy analog, only 1 reaction mixture is required
- Fluorescent labels are considered good because it eliminates the use of radioactive reagents and can be readily automated.
Sorry, I know I made that a little more painful – the best advice I can actually give is to go to the biochemistry website (www.whfreeman.com/biochem5, click on the animated techniques section, and click on chapter 6, dideoxy sequencing of DNA – after watching that (it’s a quicktime video), everything should be crystal clear)
DNA Amplification – The Polymerase Chain Reaction
- Founded by Kary Mullis in 1984
- Method for cloning a sequence within a long strand of DNA
- The sequence that is to be copied doesn’t need to be known – only the sequences flanking the target sequence must be known (so that a proper primer can be made and attached to that site)
- PCR is carried out in cycles, with exponential growth after each cycle
- In preparation, one prepares a tube that contains the template DNA, a set of primers that flank the target DNA, Taq Polymerase (Taq polymerase is used because it is heat resistant), nucleotides (lots of them so you don’t run out), and magnesium (Dr. Hampson said polymerase requires magnesium to do its job)
- Step 1 – Strand separation: The solution is heated to 95ºC to separate the parent DNA molecule into 2 strands
-
- These three steps can be carried out repeatedly by changing the temperature of the mixture
- No reagents need to be added after the first cycle
- Amplification is one millionfold after 20 cycles, and 1 billionfold after 30 cycles
- Remember, the sequence to be amplified does not need to be known – only flanking sequences
- The target sequence can be much larger than the primers (Targets > 10kb have been amplified)
- Primers do not have to be perfectly matched to flanking sequences to amplify targets. With the use of primers derived from a gene of known sequence, it is possible to search for variations on the theme. Families of genes are being discovered this way by PCR (I don’t really get this point, but it’s in the text)
- PCR is highly specific because of the stringency of hybridization at high temperatures. Stringency refers to specificity/exactness, and the required closeness of the match between primer and target can be controlled by temperature and salt.
- PCR is exquisitely sensitive – a single DNA molecule can be amplified and detected
- PCR is also a powerful technique in medical diagnostics, forensics, and molecular evolution as it provides a means of quick amplification (which then can be used to look for specific things, such as polymorphisms, or parentage (through amplification of different genes)
The Formation of Recombinant DNA Molecules – Restriction Enzymes and DNA Ligase as Tools
- A DNA fragment of interest is covalently joined to a DNA vector
- One of the most important properties of a vector is that it can replicate autonomously (Dr. Hampson uses the term epichromosomally) in an appropriate host – that is, vector DNA replicates independently of host DNA
- The vector is prepared for accepting a new DNA fragment by cleaving it at a single specific site with a restriction enzyme. These restriction enzymes usually produce staggered cuts (complementary single stranded ends) – these are known as cohesive or sticky ends because they have a specific affinity for each other
- Thus, any DNA fragment can be inserted into this plasmid if it has the same sticky ends, and this is simply done by taking a larger piece of DNA and applying the same restriction enzyme to it as was applied to the vector (plasmid) DNA
- The DNA is then inserted into the plasmid vector and DNA ligase joins the two together, forming a recombinant DNA molecule. The chains joined by ligase must be in a double helix, and an energy source (ie ATP, NAD+) is required
-
- The use of a short, chemically synthesized DNA linker – first, the linker is covalently joined to the ends of a DNA fragment or vector. In this example, the 5’ end of a decameric linker and a DNA molecule are phosphorylated and joined by ligase from T4 phage
- This ligase forms a covalent bond between blunt-ended (flush ended / non sticky-end) double-helical DNA molecules
- Cohesive ends are produced when these terminal extensions are cut by an appropriate restriction enzyme
- Thus, sticky ends corresponding to a particular restriction enzyme can be added to virtually any DNA molecule (because flush end is ligated)
- Therefore, when using a linker, the linker itself is cleaved by the restriction enzyme (the DNA fragment is not). Otherwise, the restriction enzyme is applied to the DNA fragment itself
Types of Vectors
Plasmids
- circular, duplex DNA molecules
- occur naturally in some bacteria
- range in size from 2 to several hundred kilobases (Dr. Hampson said that ideal size is between 5-10 kilobases)
- they can carry genes for inactivation of antibiotics, production of toxins, and the breakdown of natural products
- they are accessory chromosomes, and can replicate independently of the host chromosome – in Dr. Hampson’s terms, they reproduce epichromosomally
- Not all bacteria have plasmids, but some can house up to 20 of them
- An example of a plasmid is the pBR322 plasmid, which is very useful in cloning
- pBR322 contains genes for resistance to tetracycline and ampicillin
- different restriction enzymes can cleave pBR322 at a variety of unique sites at which DNA fragments can be inserted
- Insertion of DNA at EcoRI restriction site does not alter any genes for antibiotic resistance
- Insertion at the HindIII location, however, inactivates the gene for tetracycline resistance – this is an effect known as insertional inactivation. Cells containing pBR322 with a DNA insert at this restriction site are resistant to ampicillin still, but are sensitive to tetracycline – thus, they can be readily selected
- Cells that failed to take up a vector are sensitive to both tetracycline and ampicillin (since they don’t carry vector that has antibiotic resistance)
- Cells that contain the vector without a DNA insert, or a DNA insert at the EcoRI location are resistant to both ampicillin and tetracycline
- This notion of selectivity is important, as it allows scientists to easily get rid of bacteria cells they do not want (ie those without recombinant DNA vector) through the use of antibiotics
Lambda (l) Phage (Bacteriophage)
- bacteriophages are great at infecting bacteria, and the l phage, like most phages, can enjoy 2 possible lifestyles:
- The lytic pathway, where viral functions are fully expressed – viral DNA and proteins are quickly produced and packaged into virus particles. This leads to the lysis (destruction) of the host cell and the appearance of about 100 progeny virus particles (virions)
- The lysogenic pathway, where the phage DNA becomes inserted into the host-cell genome and can be replicated with host-cell DNA for many generations