PLNT 3140 Introductory Cytogenetics - 2023

Recombinant DNA II

Ligation joins two DNA fragments into a single fragment

DNA ligases are enzymes that are capable of joining different pieces of DNA together. You may remember that DNA ligases play a role in DNA replication, by joining the Okazaki fragments of lagging strand synthesis together.

We discussed in the last reading that EcoRI produces "sticky ends", or ends that have an overhang and can spontaneously base pair again. When compatible ends are in close proximity, DNA ligase can create phosphodiester bonds between the 5' and 3' ends of two fragments, re-creating the restriction site.

There are also restriction endonucleases that produce "blunt ends", like SmaI, which has the recognition sequence CCCGGG.

5' CCCGGG 3'
3' GGGCCC 5'

digest with SmaI

5' CCC 3'    5' GGG 3'
3' GGG 5'    3' CCC 5'

Not all restriction endonucleases create fragments with complementary ends, and sometime different fragments can create complementary ends for each other. The takeaway from all this is that sometimes ends can spontaneously base-pair again and sometimes not. However, base pairing is not enough for the DNA strands to be whole again. DNA ligase has to seal the sugar-phosphate backbone together.

DNA fragments are cloned into self-replicating elements called plasmids

Restriction endonucleases and DNA ligases give us the necessary means to clone genetic sequences from almost any organism into a plasmid vector. Here's how it works: We digest both the target DNA and the plasmid with the same restriction endonuclease. Let's say we used SalI. We're going to end up with overhang, "sticky" ends, which will automatically re-anneal. At many of the sites, the plasmid DNA will re-anneal with itself, but some will re-anneal with the other genetic material. What we end up with is plasmids that contain a portion of our target DNA. Usually, the plasmids are manipulated in other ways so that we can tell which plasmids have the target DNA.

A typical cloning experiment follows this outline:

1. Digest plasmid with restriction enzyme
2. Digest target DNA with a restriction enzyme that gives compatible ends
3. Ligate plasmid and insert DNA together
4. Transform bacterial host with ligation mix
5. Select for transformants with antibiotic
6. Screen for presence of an insert
7. Extract DNA from cells derived from a single, isolated colony (these are clones!)
8. Verify the identity of insert with restriction digest

Example: Cloning fragments into SalI site (G^TCGAC)
 

Clones that survive must have the plasmid, because they are ampicillin resistant. White colonies have lost β-glactosidase activity, indicating that a fragment of target DNA has inserted within the lacZ coding sequence. Therefore, we pick white colonies, each of which is a presumptive clone containing an insert.

Bluescript KS multiple cloning site (MCS)

A synthetic DNA sequence has been inserted downstream from the translation start site for the lacZ gene. Translation begins at position 816 with the ATG codon. The multiple cloning site, going from KpnI to SacI, is designed to have many convenient restriction sites, making it possible to clone a restriction fragment generated by almost any restriction enzyme somewhere in the MCS. The inserted fragment has a length divisible by 3, and sequences have been chosen so that there are no stop codons within the MCS. Although the lacZ protein will contain a few extra amino acids in its N-terminal region, these extra amino acids do not interfere with its enzymatic activity.

The sequence of the multiple cloning region shows that the lacZ gene in the Bluescript vector has been engineered to contain artificially-introduced restriction sites, to give us a choice of sites for insertion of fragments.

      m13 reverse primer: aacagct
       841       831       821         
GTGAGCGGATAACAATTTCACACAGGAAACAGCT 
CACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGA

lacZ gene ---------------->                                               KpnI                  XhoI
atg acc atg---> 802                 787                 772                 757                 742 
ATG ACC ATG ATT ACG CCA AGC TCG AAA TTA ACC CTC ACT AAA GGG AAC AAA AGC TGG GTA CCG GGC CCC CCC TCG 
TAC TGG TAC TAA TGC GGT TCG AGC TTT AAT TGG GAG TGA TTT CCC TTG TTT TCG ACC CAT GGC CCG GGG GGG AGC 
MET Thr MET Ile Thr Pro Ser Ser Lys Leu Thr Leu Thr Lys Gly Asn Lys Ser Trp Val Pro Gly Pro Pro Ser 

  SalI        ClaI    HindIII EcoRV   EcoRI   PstI    SmaI      BamHI SpeI    XbaI      NotI
                727                 712                 697                 682                 667 
AGG TCG ACG GTA TCG ATA AGC TTG ATA TCG AAT TCC TGC AGC CCG GGG GAT CCA CTA GTT CTA GAG CGG CCG CCA 
TCC AGC TGC CAT AGC TAT TCG AAC TAT AGC TTA AGG ACG TCG GGC CCC CTA GGT GAT CAA GAT CTC GCC GGC GGT 
Arg Ser Thr Val Ser Ile Ser Leu Ile Ser Asn Ser Cys Ser Pro Gly Asp Pro Leu Val Leu Glu Arg Pro Pro 

SacII     SacI
                652                 637                 622                 607 
CCG CGG TGG AGC TCC AAT TCG CCC TAT AGT GAG TCG TAT TAC AAT TCA CTG GCC GTC GTT TTA CAA C
GGC GCC ACC TCG AGG TTA AGC GGG ATA TCA CTC AGC ATA ATG TTA AGT GAC CGG CAG CAA AAT GTT G
Pro Arg Trp Ser Ser Asn Ser Pro Tyr Ser Glu Ser Tyr Tyr Asn Ser Leu Ala Val Val Leu Gln 
                                                         <--- t gac cgg cag caa aat g :m13 -20 primer

The multiple cloning site was carefully crafted so that no stop codons were inserted, and that the reading frame of the lacZ gene was conserved. The result is that the N-terminus of the β-glactosidase protein has some additional amino acids at its N-terminus. However, the enzyme is still functional even with these additional amino acids. X-GAL CLEAVAGE BY beta-GALACTOSIDASE X-gal : 5-bromo-4-chloro-3indolyl-beta-D-galactoside.

V. LABELING DNA

Labelling DNA is done by incorporating labelled nucleotides during synthesis

We've already seen with FISH that you can detect a DNA sequence on a chromosome by hybridizing a labeled DNA probe to chromsomes on a microscope slide. For identifying DNA from a gel, the DNA is first blotted onto a nylon membrane, and incubated with a labeled DNA probe. (We'll discuss gel blotting below). Probaby the most common method for making labelled probes is random-primed synthesis. This method works by denaturing DNA in the presence of random oligonucleotides.  The population of oligos includes thousands of different sequences, typically of 6 nt in length. By random chance, these hexamers will be able to hybridize many places on the template DNA. The oligos are extended by the DNA polymerase Klenow fragment, incorporating the tagged nucleotides into the newly-synthesized strands. DNA polymerases require a primer-template pair with a 3' recessed end. DNA polymerases extend 3' ends by adding nucleotides to the primer to create a double-stranded duplex. 5'---GGCT 3'---CCGAGGCTGCCTTAA                 | DNA polymerase                 V dNTPs 5'---GGCTCCGACGGAATT 3'---CCGAGGCTGCCTTAA

Another method for making labelled probes uses PCR. PCR, or the polymerase chain reaction, is a method for amplifying segments of DNA [PCR Review]. The DNA labelling reaction using PCR is similar to the regular PCR reaction, except labelled nucleotides are incorporated with the unlabelled nucleotides. Some advantages of choosing to label DNA this way are:

• Only very small (ng) amounts of template are required
• Saves having to isolate inserts and label separately
• Eliminates virtually all vector contamination in probes

Labeled nucleotides can be detected by a number of different methods

Although radioisotopes were used for probe synthesis at one time, radioisotopes have been replaced by two general approaches to non-radioactive labeling. Both of these methods incorporate various nucleotide analogs into DNA in a labeling reaction. In chemiluminescence, an enzyme is conjugated to a molecule such as an antibody that is specific for the nucleotide analog. When the appropriate substrate is added, the enzyme breaks it down, resulting in the emission of a photon of light. In fluorescent detection, nucleotide analogs themselves contain a fluorescent tag, which emits light when illuminated with the appropriate excitation wavelength. Commonly used nucleotide analogs include: Dioxygenin-labelled nucleotides (DIG-dNTPs), biotin-labelled nucleotides, and fluorescent nucleotides. Each of these types of nucleotide analogs is detected differently.

Name	Detection Method	Example
DIG-dNTPs	Detection of DIG-labeled nucleotides is done using anti-DIG antibodies conjugated to alkaline phosphatase. When a substrate such as CDP* is added, alkaline phosphatase breaks down the substrate, which emits a photon of light.
Biotinylated DNA	Detection of biotinylated DNA is typically done using streptavidin, a bacterial toxin that has an affinity for biotin, conjugated to horseradish peroxidase. Breakdown of substrate (eg. luminol peroxide from Clontech Inc.) results in release of a photon of light.
Fluorescent DNA	Fluorescently-tagged DNA probes have the advantage that no enzymatic reaction is necessary for detection. Dye-conjugated nucleotides are incorporated into DNA during labeling eg. PCR labeling.

Hybridization of labeled DNA to target DNA can detect genes, RNA, etc.

The purpose of hybridization is to use a labelled sequence of DNA (called a probe) to detect a specific, complementary sequence in a genome. Hybridization has many uses, including:

• Finding a whole gene when you have a fragment
• Seeing if a gene in one species is present in another species
• Detecting the amount of RNA in a cell

The image at right is a representation of the filter (grey) used in hybridization experiments. Once the unlabelled DNA is denatured (with an alkaline solution like NaOH), the transfer is made at neutral pH to keep the DNA mostly in single-stranded form. The DNA then adheres to the filter strands. The probe is denatured as well, and combined with the filter to incubate. Then, the probe is free to base pair with complementary regions. After sufficient time has passed, washing is used to clean excess unhybridized probe from the filter. On the filter, there is little opportunity for complementary strands to find each other, so most DNA remains single-stranded.

By blotting the DNA from the gel to a nylon filter, the DNA bands are transferred in the same pattern in which they occur on the gel. Now, if  we incubate the filter with the labeled DNA probe, the probe will bind to only those bands that have a sequence complementary to the probe. Even imperfect matches between the probe and the target DNA will result in a stable duplex, which we detect as signal on the filter.

Detection is done either using fluorescence or chemiluminescence, as described previously.

See:  SCHEMATIC OF HYBRIDIZATION EXPERIMENT

Example

Transgenic rescue of hemolytic anemia due to red blood cell pyruvate kinase deficiency
Hitoshi Kanno, Taiju Utsugisawa, Shin Aizawa, Tsutomu Koizumi, Ken-ichi Aisaki, Takako Hamada, Hiromi Ogura, Hisaichi Fujii
Haematologica June 2007 92: 731-737; doi:10.3324/haematol.10945

Mice deficient for the expression of pyruvate kinase in red blood cells (R-PK) were were transformed with a human gene for R-PK.  A southern blot was used to test segregating progeny, to see which progeny had the gene, and to compare copy numbers of the inserted gene.

Procecure:
1. Total DNA was extracted from blood cells taken from individual mice.
2. Total DNA was digested with BamHI.
3. DNA was run on agarose gels containing ethidium bromide, and gels visualized in UV light. Because the BamHI recognition sequence is only 6 bases long, it should occur hundreds of thousands of times in the mouse genome, resulting in too many bands to resolve on the EtBr stained gel. The result is a smear of DNA bands, as seen at left. The unmarked lane is a size marker, with bands of known fragment size. Lanes 1 - 6 have DNA from six different mice.
4. DNA from the gel was transferred by Southern blotting to a nylon membrane.
5. The blot was hybridized with the human R-PK gene which had been labeled with DIG chemiluminescent label. The chemiluminescent image of the blot is shown at right. The intensity of the bands roughly indicates the relative number of copies of the gene inserted into mouse chromosomes. Note that mice 3 and 5 did not receive any copies of the R-PK gene.

DNA Sequencing

DNA sequencing methods have come a long way since the early 1990s. The methods below are all "next generation sequencing" or NGS methods. The key feature of NGS methods is that they are able to sequence thousands or millions of short DNA fragments at a time, drastically reducing the per bp cost of sequencing. The types of sequencing differ depending on whether you need to sequence an unknown genome (de novo), or resequence an already-known genome.

All of these methods share a common strategy:

1. Cut genomic DNA into millions of short fragments
2. Sequence millions of fragments
3. Use genome assembly software to put together overlapping sequencing reads into contigs. Contigs are large fragments of chromosomes

Pyrosequencing

Pyrosequencing uses pyrophosphate as a indicator. When a dNTP is added, pyrophosphate is released and through a cascade, cleaves a substrate called luciferin, which then emits a photon. Only 1 of the 4 dNTPs is only added in each cycle, so if a light signal is detected you know which nucleotide was added. This process produces reads of around 500 bp.

Original:

Illumina

Illumina sequencing uses dNTPs that all fluoresce at 4 different wavelengths. Fragments of DNA are tethered to a flowcell, and amplified so that many copies of each fragment can be used. Then, when each nucleotide is added, that particular area on the flowcell flashes a distinctive colour for each different nucleotide. Current Illumina platforms can read up to 250 bases per read.

Single Molecule Real-Time Sequencing

Single molecule real-time sequencing, or SMRT, is a technology developed by the company Pacific Biotechnology. This technology also uses differently-labelled nucleotides, but they are attached to the terminal phosphate instead of to the sugar. SMRT uses an extremely small detection chamber to catch the flashes of different coloured light to read the sequence of the DNA. The advantage of this method is that it produces much longer read lengths, to about 20,000 bp. However, it has a high error rate.

These technologies all produce sequences, or "reads", only of a certain length. In order to assemble these reads into a continuous genome, we need different tools.