Molecular biology week 3A - Nucleic acid chemistry
Factors that determine Tm: (melting point
- DNA concentration - DNA length - Base pair composition (higher content of G-C pairs = higher Tm) - Concentration of cations in the buffer (Mg2+, K+)
Hybridisation-based techniques
- DNA denaturation/renaturation based techniques: DNA composition and complexity analysed in DNA solutions - Analysis of hyperchromic effect - Analysis of reassociation kinetics - In situ DNA hybridisation: DNA presence and localisation / organisation. - In situ RNA hybridisation: Gene expression (Levels of gen transcription)
Hybridisation-based techniques
- DNA denaturation/renaturation based techniques: DNA composition and complexity analysed in DNA solutions - Analysis of hyperchromic effect - Analysis of reassociation kinetics In situ DNA hybridisation: DNA presence and localisation/organisation. - In situ RNA hybridisation: Gene expression (transcription)
Hybridisation based techniques
- In situ DNA hybridisation: DNA localisation - In situ RNA hybridisation: DNA expression
In situ RNA hybridisation
- In situ RNA hybridisation: DNA (gene) expression
Nucleic acid chemistry
- Nucleotides are linked by a phosphodiester bond between the phosphate group at the C-5' position and the OH group on the C-3' position -There is a linkage of two nucleotides by the formation of a C-3'-C-5' (3'-5') phosphodiester bond producing a dinucleotide -Shorthand notation for a polynucleotide chain
Sanger Method - The principle
- Synthesis of truncated daugther strands is accomplished by use of 2'-3'-dideoxyribonucleoside triphosphates (ddNTPs) labelled with 32P - These molecules (ddNTPs), in contrast to normal deoxyribonucleotides (dNTPs), lack a 3'- hydroxil group
Capillary electrophoresis
-A method of separating DNA samples based on the rate of movement of each component through a gel-filled capillary while under the influence of an electric field -A very limited amount of nucleic acid, 500-1500 bp in size, needs to be analyzed in a short time (same day) with the results available immediately. Capilary electrophoresis will satisfy those conditions. -The detector is linked to a computer, Generates automated output with a file containing DNA sequence
Analytical techniques to investigate of DNA and RNA
-Absorbance: Nucleic acids absorb UV light most strongly at 260 nm due to interaction between UV light and the ring systems of the bases -In molecular hybridization DNA strands (or DNA and RNA strands) can be renatured to each other. This has provided the basis for several techniques for analysing DNA
The reaction: DNA polymerisation
-Actually 4 separate reactions (4 separate tubes) -Each reaction/tube with a different labelled ddNTP Each tube contains -Template DNA (to be sequenced) -dATP, dCTP, dTTP, dGTP -One of the four ddNTPs labelled with 32P, at low concentration -Oligo primer -DNA polymerase
THE STRUCTURE OF DNA: THE DOUBLE HELIX
-DNA consists of two polynucleotide chains wound around each other in the form of a right-handed double helix -The double helix has a major and a minor groove -The two polynucleotide strands are anti-parallel (one is in 5'-3' orientation and the other is in 3'-5' orientation) The sugar-phosphate backbones are on the outside, the bases on the inside -The two strands are held together by hydrogen bonds between the bases
Sanger method
-DNA is made in the lab, but some of the bases are altered, and cause the growing DNA chain to end at that base. These altered bases are dideoxynucleotides (ddNTPs), so this method is also called dideoxy sequencing. -A technique used to determine the original sequence of a DNA where dideonucleotides (which lack a 3 prime hydroxyl and thus halt replication wherever they attach) are added to the mix along with everything else needed for replication (DNA polymerase, primer, RNA polymerase, DNA nucleic bases, RNA nucleic bases) to a tube; when the dd binds it halts replication and creates fragments at pretty much every point possible; when a gel is run the sequence of the DNA can thus be determined
Generation of genetically modified cells
-Expression of exogenous proteins to study dynamics of cell adhesion and cytoskeletal proteins -Generation and expression of fluorescent fusion proteins in cells
Maxam-Gilbert
-First sequencing method, 4 tubes with 4 reactions. The reactions cut at G, G+A, C+T, and C. Done by first methylating the pair in question with DMS and then cleaving with alkali. Then doing electrophoresis and comparing the bands to determine sequence. -Helps to find the chemistry of a DNA sequence by cleavage. - Single stranded DNA fragment that was separated through gel electrophoresis is labeled radioactively on one end and divided into four tubes; DNA backbone is selectively cut once at G, G+A, C+T, and C using chemicals in a controlled reaction
In situ DNA hybridisation (Fluorescent in situ hybridization)
-Fluorescent in situ hybridization (FISH) is used to identify the chromosomal location of a DNA of interest - e.g. Fluorescent in situ hybridization (FISH) of human metaphase chromosomes. The probe specific to centromeric DNA, produces a yellow fluorecence signal indicating hybridization. The red fluorescence is produce by propidium iodide (DNA intercalating agent) counterstaining of chromosomal DNA.
DNA digestion
-Fragments resulting from restriction enzyme digestion: -Blunt end: A fragment of DNA resulting from the breaking of DNA molecule in which there are no unpaired bases or overhangs in the end, hence, both strands are of the same length. -Sticky end: A fragment of DNA in which the terminal portion has a stretch of unpaired nucleotides, and the strands are not of the same length.
DNA electrophoresis
-Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge. -Agrose gel has pores which affect movement of small and large fragments (large fragments move more slowly and travel less distance than the smaller fragments)
Hybridisation
-Hybridization is the process of combining two complementary single-stranded DNA or RNA molecules and allowing them to form a single double-stranded molecule through base pairing. Molecular hybridization is possible between: -Two DNA strands -One DNA and one RNA strand
Hybridization techniques
-Hybridization probes can be used to detect the presence of their complementary sequence in a number of hybridization applications (Table 1). -Techniques can be used in conjunction with genomic sequence data to measure gene expression?
Hybridisation
-Hybridization: The process of combining 2 complementary single-stranded DNA or RNA molecules and allowing them to form a single double-stranded molecule through base pairing. - Enhanced absorbance of denatured DNA vs native DNA (folded DNA structure) - Allows for HYBRIDISATION
Molecular hybridization techniques Hyperchromic effect: Analysis of DNA complexity
-Hyperchromic effect: An increase in absorption at a particular wavelength of light by a solution or substance due to structural changes in a molecule -Melting temperature or melting point (Tm): Temperature in degrees Celsius, at which 50% of all molecules of a given DNA sequence are hybridized into a double strand, and 50% are present as single strands
Automated output
-In an automated sequencing equipment, the four reaction mixtures are subjected to gel electrophoresis and the order of appearance of each of the four different fluorescent dyes at the end of the gel is recorded -The sequence of the original template DNA can be read directly (N= nucleotide that cannot be assigned)
In situ RNA hybridisation
-In situ RNA hybridisation: DNA (gene) expression: Use of DNA labelled probes (fluorescence, digoxigenin, radioisotopes) -Fluorescence-labelled/tagged probe for RNA hybridisation
DNA digestion
-Restriction enzymes (endonucleases) recognise palindromic sequences -A palindromic sequence is a nucleic acid sequence on double-stranded DNA (or RNA) wherein reading 5' (five-prime) to 3' (three prime) forward on one strand matches the sequence reading backward 5' to 3' on the complementary strand with which it forms a double helix.
Oligo primer
-Short strand of RNA or DNA (generally about 10 base pairs) that serves as a starting point for DNA synthesis. It is required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA. DNA polymerase.
Sanger Method
-The basic idea behind this method is to synthesize from the DNA fragment to be sequenced a set of daughter strands that are labelled at one end and differ in length by one nucleotide -Reaction: DNA synthesis/polymerisation -The reaction mix: -Template strand -Non-labelled nucleotides -Type of ddNTPs (2',3'-Dideoxynucleotide triphosphates) -Primer -DNA polymerase III
Electrophoresis
-The movement of suspended particles through a fluid or gel under the action of an electromotive force applied to electrodes in contact with the suspension. - Electrophoresis is the basis for several analytical techniques -Electrophoresis separates the molecules in a mixture by causing them to migrate under the influence of an electric field (polinucleotides are negatively charged, based on the phosphate groups, nucleotides chain move to the positively charged pole) - Nucleic acid electrophoresis separates DNA and RNA fragments by size such that smaller fragments migrate at a faster rate through a gel than large fragments
More questions
-What is a DNA intercalating agent? -Name the intercalating agents mentioned during the lecture and their common applications. - According to the radiography below of a gel electrophoresis from a Sanger reaction, what is the sequence of the DNA fragment under study?
Modifications to the sanger method technique
-ddNTPs are fluorescence-tagged instead of 32P -ddNTPs are fluorescence-tagged -Relies on capillary electrophoresis -Samples loaded at one end of a tube -As they emerge, they pass over a fluorescence detector -Allows some automation
Answer these questions
1. Write sequence of complementary 3'-5' strand of the following 5'-3' DNA sequences a) GCGCGGGCTA; b) GCCAATTCAT; c) TATACGAATT 2. Write the complementary mRNA sequence of the coding DNA sequences above. 3. Order these DNA sequences according to melting point (Tm) value (from low to high).
Answers
1. a) GCGCGGGCTA; b) GCCAATTCAT; c) TATACGAATT a) CGCGCCCGAT; b) CGGTTAAGTA; c) ATATGCTTAA 2. a) CGCGCCCGAU; b) CGGUUAAGUA; c) AUAUGCUUAA 3. c), b), a)
Principles of DNA Sequencing -Introduction
1.In each tube, DNA fragment to be sequenced + DNA processing compounds 2. Reaction 3. Processed DNA mixtures (labelled DNA fragments) 4. Separation of DNA fragments by electrophoresis
Hybridization techniques
1.Southern blot: DNA fragments separated by gel electrophoresis 2.Northern blot:RNA fragments separated by gel electrophoresis 3.Slot/dot blot:Total DNA or RNA 4. Colony blot: DNA or RNA in microbial colonies 5.Fluorescent in situ hybridization (FISH): DNA or RNA in microbial cells 6.Microarray:DNA which is hybridized to probes on an array 7. Quantitative PCR:DNA fragments during PCR amplification
In situ hybridization (ISH)
A type of hybridization that uses a labeled complementary DNA, RNA or modified nucleic acids strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough (e.g., plant seeds, Drosophila embryos), in the entire tissue (whole mount ISH), in cells, and in circulating tumor cells (CTCs). This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections.
Process of in situ hybridisation
A typical target-specific probe will contain 40 oligonucleotides, resulting in 20 oligo pairs that bind side-by-side on the target for detection of mRNA and lncRNA, and 2 oligos or a single pair for miRNA detection. Signal amplification is achieved via a series of sequential hybridization steps. A pre-amplifier molecule hybridizes to each oligo pair on the target-specific RNA, then multiple amplifier molecules hybridize to each pre-amplifier.
Process of in situ hybridisation
An alternative technology, branched DNA assay, can be used for RNA (mRNA, lncRNA, and miRNA ) in situ hybridization assays with single molecule sensitivity without the use of radioactivity. This approach (e.g., ViewRNA assays) can be used to visualize up to four targets in one assay, and it uses patented probe design and bDNA signal amplification to generate sensitive and specific signals. Samples (cells, tissues, and CTCs) are fixed, then treated to allow RNA target accessibility (RNA un-masking). Target-specific probes hybridize to each target RNA. Subsequent signal amplification is predicated on specific hybridization of adjacent probes (individual oligonucleotides [oligos] that bind side by side on RNA targets).
Sanger method - dideoxy method
DNA polymerase connects adjacent deoxynucleotides by covalently linking the 5' P of one to the 3' OH of another. Nucleotides missing the 3' OH can be synthesized. They are called ddNTPs
Initiation of DNA polymerisation requires priming
DNA polymerisation during replication in a cell. The replication fork
DNA digestion
DNAases (Deoxyribonucleases): Enzymes that hydrolize the phosphodiester bonds in the DNA backbone leading to dissociation of the adjacent nucleotides. Two types: 1. Endonucleases: DNAses that cleave DNA within a polynucleotide chain. 2. Exonucleases: DNases that cleave only nucleotides at the ends of DNA molecules
Nucleic acids can be separated using:
Electrophoresis!
RNases (Ribonucleases)
Enzymes that hydrolize the phosphodiester bonds in the RNA backbone leading to dissociation of the adjacent nucleotides.
Process of in situ hybridisation
For hybridization histochemistry, sample cells and tissues are usually treated to fix the target transcripts in place and to increase access of the probe. As noted above, the probe is either a labeled complementary DNA or, now most commonly, a complementary RNA (riboprobe). The probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away (after prior hydrolysis using RNase in the case of unhybridized, excess RNA probe). Solution parameters such as temperature, salt, and/or detergent concentration can be manipulated to remove any non-identical interactions (i.e., only exact sequence matches will remain bound).
Molecular hybridization techniques Hyperchromic effect: Analysis of DNA complexity
Hyperchromic effect: An increase in absorption at a particular wavelength of light (260 nm) by a solution or substance due to structural changes in a molecule (DNA)
In situ hybridization (ISH)
In situ hybridization is a powerful technique for identifying specific mRNA species within individual cells in tissue sections, providing insights into physiological processes and disease pathogenesis. However, in situ hybridization requires that many steps be taken with precise optimization for each tissue examined and for each probe used. In order to preserve the target mRNA within tissues, it is often required that crosslinking fixatives (such as formaldehyde) be used.
In situ hybridization (ISH)
In situ hybridization is used to reveal the location of specific nucleic acid sequences on chromosomes or in tissues, a crucial step for understanding the organization, regulation, and function of genes. The key techniques currently in use include: in situ hybridization to mRNA with oligonucleotide and RNA probes (both radio-labelled and hapten-labelled); analysis with light and electron microscopes; whole mount in situ hybridization; double detection of RNAs and RNA plus protein; and fluorescent in situ hybridization to detect chromosomal sequences. DNA ISH can be used to determine the structure of chromosomes.
The key to separation is based on the:
Matrix (pores) of the gel: -Polyacrylamide gel -Agarose gel
Process of in situ hybridisation
Next, multiple label probe oligonucleotides (conjugated to alkaline phosphatase or directly to fluorophores) hybridize to each amplifier molecule. A fully assembled signal amplification structure "Tree" has 400 binding sites for the label probes. When all target-specific probes bind to the target mRNA transcript, an 8,000 fold signal amplification occurs for that one transcript. Separate but compatible signal amplification systems enable the multiplex assays. The signal can be visualized using a fluorescence or brightfield microscope.
- Electrophoresis-based and transfer to a membrane ("blot"):
Northern blot (RNA) Southern blot (DNA)
Southern and Northern blot hybridization
Southern blot hybridization refers to the detection of specific DNA fragments that have been separated by gel electrophoresis (Figure 1). After the electrophoresis the separated DNA fragments are denaturated and transferred to a nitrocellulose (or nylon) membrane sheet by blotting. In the blotting the gel is supported on a sponge in a bath of alkali solution, and buffer is sucked through the gel and the sheet by paper towels stacked on top of the nitrocellulose sheet. The buffer denaturates the DNA and transfers the single stranded fragments from the gel to the surface of the sheet, where they adhere firmly. The nitrocellulose sheet containing the bound single-stranded DNA fragments is pealed off the gel and placed in a sealed plastic bag or a box together with buffer containing labelled DNA probe specific for the target DNA sequence. The sheet is exposed to the probe under conditions favouring hybridization. After the hybridization, the sheet is removed from the bag, washed thoroughly to remove unhybridized probes and viewed using autoradiography or ultraviolet light depending on the labels used (radioactive of fluorescent). An adaptation of Southern blotting is Northern blotting, in which RNA molecules are electrophoresed through the gel instead of DNA.
Principles of DNA Sequencing Introduction
The complete characterization of any DNA fragment of interest requires determination of its nucleotide sequence
Process of in situ hybridisation
Then, the probe that was labeled with either radio-, fluorescent- or antigen-labeled bases (e.g., digoxigenin) is localized and quantified in the tissue using either autoradiography, fluorescence microscopy, or immunohistochemistry, respectively. ISH can also use two or more probes, labeled with radioactivity or the other non-radioactive labels, to simultaneously detect two or more transcripts.
Northern Blot
Transfer of electrophoretically separated RNA molecules from a gel onto an absorbent sheet, which is then immersed in a labelled DNA probe that will hybridize to an RNA of interest to reveal its presence.
Southern Blot
Transfer of electrophoretically separated fragments of DNA, from the gel to an absorbent sheet of material, such as nitrocellulose, to which the DNA binds. Once DNA is transferred on the sheet, this is immersed in a solution containing a labelled DNA probe that will hybridize to fragment(s) of interest.
Principles of DNA Sequencing- Introduction
Two methods developed approximately at the same time: - Maxam Gilbert method - Sanger method
Learning Objectives
Understand: -Molecular hybridisation-based techniques -DNA electrophoresis, Southern and Northern blot -DNA sequencing
Exam question 2013-2014
Using as source of biological material cells, tissues or isolated RNA, what molecular biology techniques could you use to detect gene expression? Name and explain the principle and methodology.
Werner Arber, Daniel Nathans, Hamilton O. Smith
Won a nobel peace prize in 1978 for discovering restriction enzymes and their application to problems of molecular genetics in 1970.
