Bioc 2001 - Summary Molecular Biology PDF

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BIOC 2001 Nucleic acids Professor Shepard The central dogma for molecular biology DNA makes RNA makes proteins. DNA bases A pairs with T, C pairs with G. Purines (larger) are guanine and adenine. Pyrimindes (smaller) are cytosine and thymine. Purines are two ringed structures, whereas pyrimidines ar...

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BIOC 2001

Nucleic acids Professor Shepard The central dogma for molecular biology - DNA makes RNA makes proteins. DNA bases - A pairs with T, C pairs with G. - Purines (larger) are guanine and adenine. Pyrimindes (smaller) are cytosine and thymine. - Purines are two ringed structures, whereas pyrimidines are one-ringed structures. - Carbons in are labelled 1 to 5 with ‘. - Sugar in DNA is deoxyribose, lacking the 2’ OH and instead only has a hydrogen atom on the 2’ carbon.

- The 3’ OH group is required for phosphodiester bond formation with adjacent bases. Nucleosides - Nucleosides are bases (A,T,C,G) and sugar (deoxyribose). - In nucleosides, the bases have different names: adenosine, thymidine, guanosine, cytidine. The nucleosides (base and sugar) are therefore called deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine. Nucleotides - Composed of base, sugar and phosphate, i.e. a nucleoside with a phosphate attached. - The building blocks of DNA have three phosphate groups (alpha, beta, gamma) - Deoxynucleoside triphosphates (DNA building blocks) i) dATP = deoxyadenosine-5’-triphosphate ii) dGTP = deoxyguanosine-5’-triphosphate iii) dCTP = deoxycytidine-5’-triphosphate iv) dTTP = deoxythymidine-5’-triophosphate. NB dNTP = nucleotide.

- DNA chain (polynucleotide chain) has polarity; the first building block always has three phosphates but when joined two phosphates are released as inorganic phosphate (beta and gamma), Pi. - Alpha remains, and hence the first DNA base will confer a negative charge as a phosphate group.

- The 5’ end of DNA chain is different to the 3’ because 5’ has a phosphate group whereas the 3’ end has an OH on the 3’ carbon whilst the 5’. Chargaff’s rule - In DNA, the number of A base is equal to the number of T bases. Similarly, G = C. - Therfore, A binds to T and C binds to G. - Two hydrogen bonds are formed between an AT pairing, whilst 3 hydrogen bonds are formed between a GC pairing. - A purine will always bind a pyrimidine; this is important with regards to the shape of the helix. - DNA has a negative net charged because of the phosphate group at the 5’ end. - DNA strands run anti parallel to one another i.e. 5’ to 3’, and 3’ to 5’.

- There are major and minor groves on the DNA were DNA bidning proteins bind. - The GC base pairing is stronger than the AT base pairing. - Hydrogen bonding between the bases provides specificity through precise requirements. Enzymes that act on DNA - Nucleases cleave the phosphodiester bond between the DNA bases. - If you want to clone DNA, then you must first use nucleases to cut up chromosomes into much smaller, manageable fragments. - Endonucleases cleave bonds internally i.e. within the molecule - Exocnucleases cleave the bond from either the 5’ end of DNA of the 3’ end, cleaving each bond one at a time. - Type II restriction nucleases are produced by bacteria, cleave both strands of double stranded DNA at specific sequences. - Cuts can be staggered producing ‘overhands’ on both strands of DNA that can be re annealed to strands with complimentary overhangs. - Recognition sites for restriction endonucleases are palindromic. Pst 1 (Pseudomonas stuarti 1) - Pst 1 is a type II restriction endonuclease which can cut at palindromic recognition sits on the DNA, producing 3’ overhangs.

EcoR1 (E. coli) - Type II restriction endonuclease which cuts at palindromic recognition sites on DNA producing 5’ overhangs.

Haemophilus parainfluenzae 1 (Hpa 1) - Type II restriction endonuclease which cuts at palindromic sequences, producing blunt ends.

DNA ligase - It is used by the cell to catalyse the formation of the phosphodiester bond between adjacent DNA bases present in duplex DNA. - It requires a 5’ phosphate and 3’ OH. - Sticky ends of DNA are re annealed through complimentary base pairing. - Blunt end ligation takes longer to be completed by sticky end ligation. - Mechanism of DNA ligase: enzyme contains a lysine residue, where the R group is NH3+. Step 1) Adenylation of ligase on the lysine residue (by AMP from ATP or NAD+). NAD is in bacterial ligase, ATP is in eukaryotic, viruses and phase ligases.

Step 2) Activation of the 5’ phosphate at the 5’ terminus. AMP transferred to the 5’ phosphate at the 5’ terminus. An attack by the AMP molecule creates a phospodiester bond between the 5’ phosphate and the 3’ OH.

Step 3) Displacement of the bound AMP seals the nick.

Alkaline phosphatase - Calf intestinal phosphatase often used in labs - It removes the 5’ phosphate from RNA or DNA, creating an OH group instead. - As a result the RNA/DNA molecule is resistant to ligation. - It prevents plasmids cut with restriction enzymes from re annealing, which will increase the number of recombinant plasmids produced in these types of experiments. Separation of DNA strands - In our cells, DNA strands are separated by specific enzymes. - In the lab, we use both a high pH and heat to achieved this. - When DNA strands go from an ordered state to a disordered state, there is an increase in the absorbance at 260nm. - This is known as hyperchromicity.

- Tm = melting temperature when the DNA strands separate. - Tm depends on sequence composition; there are 3 hydrogen bonds in a GC base pairing whereas there are only 2 hydrogen bonds in an AT base pairing, therefore sequences with a high GC content will have a higher melting temperature. - Double stranded (ds) DNA can be separated, and later come together. - Double stranded DNA can also separate to form hybrids. - The ability of DNA strands to bind each other through complimentary base paring is a characteristic that is fundamental for PCR, microarray analysis and Northern blots hybridization. Southern Blots - DNA is broken up with enzymes and is now single stranded. It is then transferred onto a membrane. - Probes are added to the membrane, these can be genes which have been radioactively probes or fluorescently probed. The probes bind to corresponding DNA through complimentary base pairing - If a radioactive probe is used, a film is placed over the membrane. The film contains silver grains

that interact with the radioactive probe, producing an image. - These probes can also be tagged so that a colour reaction occurs when it binds to corresponding DNA on the membrane. DNA topology - Topoisomerase type I can relax DNA by introducing transient single stranded or double stranded breaks in the DNA – it knocks the phosphodiester bond - It is involved in DNA replication and prevents strands getting tangled after DNA replication. Blue white assay - Used to determine whether bacterial cells have taken up a plasmid and whether the plasmid has successfully been made recombinant. - Mutant DH5a E. coli cells are transformed. They lack the Z gene, which encodes for the alpha of the enzyme beta galactosidase. - The pUC19 used in the experiment can compliment such mutants, through hthe process of homologous recombination – sequences of homology between the plasmid and the bacterial chromosomes are required for this to happen. - The plasmid also contains genes for ampicillin resistance. - The DH5a E. coli cells are grown on agar containing ampicillin; cells which have not taken up the plasmid will not be able to grow on the plate so all colonies on the plate will contain cells that have successfully been transformed and hence contain the puC19 plasmid. - The gene fragment has been cloned at site which interrupts the lac z gene on the plasmid, therefore recombinant plasmids will not be able to complement the mutant DH5a cells. - The agar used contains the chemical X-gal, which can be cleaved by beta galactosidase to produce a blue product. - As recombinant cells which not contain a functioning lac Z gene, they will not be able to produce beta galactosidase and therefore not convert X-gal into a blue product. Instead, these colonies remain white. - Cells that are not recombinant will be able to produce beta galactosidase and therefore these colonies appear blue on the plate. - IPGT is a gratuitous inducer of the lac operon; it works by binding to the repressor and hence prevents it from binding to the lac operator so the lac operon can be expressed. Understanding our genome Professor Shepard How do we learn about DNA? - Clone DNA - Sequence DNA - Analyse sequence information Cloning DNA - Genomic libraries: the library includes clones that covers the whole genome - cDNA libraries: the library includes clones that correspond to mRNA sequences. - Cloning was carried out this way when technology was limited. Protein coding genes - Genes contain introns and exons. - When mRNA is produced during transcription, introns are removed and the exons are joined together to produce the mature mRNA transcript. - When making cDNA libraries, mRNA must be isolated from the other RNA in the cell. Oligo dT chromatography - Used to separate mRNA (2-4%) from the rest of the RNA in the cell (tRNA (6%), rRNA (90%)) - When eukaryotic mRNA is made, it is spliced, then capped and polyadnelyated to protect is from enzymes in the cell; mRNA is not very stable.

- The 3’ end of the mRNA is polyadenylated, and the 5’ end is capped. - tRNA and rRNA do not contain this feature, therefore it can be used to separate mRNA from the two. - The matrix of the column contains dTs (deoxythymidines) - It is an affinity column, mRNA which contain a poly A tail at their 3’ end will bind to the dTs in the matrix through complimentary base pairing via hydrogen bonds. rRNA and tRNA cannot bind to the column because they do not contain a poly A tail. - Any mRNA retained on the column is then washed off the matrix using a high salt, which is able to break the hydrogen bonds between the A and T base pairs. Cloning mRNA - To clone DNA from mRNA, it first needs to be copied into cDNA, because this is what plasmids and restriction enzymes work with. - mRNA is very fragile, so it needs to be treated very carefully because it degrades very rapidly. - A primer is required for cloning mRNA; an olido dT primer is used (binds to the poly A tail through complimentary base pairing and hydrogen bonds).

- The enzyme reverse transcriptase is used to convert mRNA into cDNA. - For this step, reverse transcriptase, an mRNA temple, oligo dT primer and free dNTPs are needed. - This step produces hybrid molecule composed of two strands, one strand of RNA and the other strand of complimentary cDNA.

- We want to remove the RNA strand so we can produce double stranded DNA. - This is done using the enzyme Ribonuclease H (RNase H); the H stands for hybrid. This enzyme is able to cleave the phosphodiester bonds in an RNA molecule that is bound to a DNA molecule.

- Once the RNA is removed, it must be replaced using the enzyme DNA polymerase. It uses cDNA to synthesise a complimentary cDNA strand. - DNA ligase then fills in the gaps between the newly synthesised DNA strand, and re enforces the newly formed phosphodiester bonds in the newly DNA strand.

- A molecule of double stranded DNA with no introns has been produced. - The double stranded cDNAs are cloned into vectors, such as the plasmid vector pUC19. They are then transformed into bacteria and colonies are produced. - A DNA library is a collection of clones, an each clone carries a different cDNA molecule. Cloning and sequence the Human Genome - The human genome is fragmented using restriction enzymes. - The fragments are then put back together in the correct order; this needs to be done for each chromosome.

- The chromosome is broken up into over lapping fragments, this is done by digesting the DNA with restriction enzymes; only small amounts of the restriction enzyme are added some that a number of the restriction sites on the DNA are cleaved, but not all of them. - This produces a ‘contig’, which is a continuous set of fragments, which map across the whole chromosome. DNA libraries - C means either copied or cloned. - A DNA library is a collection of bacterial clone, each clone carries a different cDNA molecule.

Bacterial artificial chromosomes (BACs) - Used to create libraries with large fragments of DNA from the chromosome. - If the fragments were too small then it would be impossible to put back together the sequences in the right order as found on the chromosome. - The inserts cloned in BACs must be around 100,000bp. - Special precautions must be taken when using BACs to sequence genomes. - The DNA needs to be protected because it is prone to mechanical sheer; this needs to be avoided as exposing the DNA to mechanical sheer may result in false results being obtained. Method for BACs 1) White blood cells are mixed with agarose and placed in a mould, which is the same size as the wells in the agarose gel used for gel electrophoresis. White blood cells are used because they can be obtained in a non-invasive way. 2) The cell wall of the white blood cells in the agarose are ruptured. 3) Restriction enzymes are added to digest the DNA in the agarose gel. 4) Each mould containing the white blood cells is then placed in the agarose gel wells. 5) The gel is run under UV light. DNA of approximately 100,000bp is then excised from the gel. This is done by removing sections of the agarose containing bands, which correspond to DNA of approximately 100,000bp. 6) The DNA from the cut sections of gel is then eluted. 7) The DNA is then ligated to a plasmid vector that has been cut with the same restriction enzymes used to cut the gel. This ensures that the ends of the DNA and the plasmid are complimentary to one another. 8) The plasmid and the DNA are then treated with ligase to ‘reseal’ the phosphodiester backbone between the nucleotides. 9) The bacteria are transformed with the plasmids; this creates a library containing large DNA fragments. 10) The transformed bacteria are then picked into 384-well plates. A grid is used so that it is known where each clone is. 11) Bacterial DNA is isolated from cells for sequencing reactions. If the sequence of the plasmid is known, then we can design a primer, which is complimentary to the sequence of the plasmid. - A primer is always needed to sequence DNA. 12) The ends of the large fragments of DNA are sequenced, and with the help of a computer these sequences are lined up to form a back bone of the chromosome scaffold, where the overlapping ends of the sequences are lined up, to produce Contigs. The Contigs are linked by sequencing the ends of the large DNA fragments.

13) The Contigs are then broken up into smaller fragments, and these are sequenced.

14) Using the shotgun approach, the smaller sequenced fragments are then used to ‘fill in’ the sequences of the larger fragments. This involves using a combination of short and long clone libraries. - This is how we build up a sequence for the genome. - Smaller libraries need a larger number of plasmids. DNA sequencing - The human genome was first sequenced using the ‘Dideoxy Chain Termination Method’. - This is known as the Sanger Method. - It uses dNTPS that lack the 3’OH; these molecules are called dideoxynucleoside triphosphate (ddNTPs)

- The sequence for the primer used is deciphered using the sequence of the plasmid. - Sequencing requires a radioactive primer, DNA polymerase enzyme and dATP, dGTP, dTTP and dCTP, as well as ddATP, ddGTP, ddUTP and ddTTP. - The four ddNTPs are placed in separate test tubes, and are added to the reaction in low concentrations. The reactions are carried out in four different testubes. - Every time when a ddUTP is incorporated, chain termination occurs as a result; a phosphodiester bond cannot be formed between the dNTP and ddNTP as the 3’ OH is required to form the bond. - Fragments of different sizes are produced as a result.

- The different sized fragments produced are then electrophoresed and separated according to size. - The position of each fragment in the gel is dependent on the position of the fragment in the sequence.

- However, this method is too time consuming and expensive; there are four different experiments going on in four different test tubes. - Instead, an automated process has been developed which uses dNTPs and a fluorescent tag. - For the automated sequence, each ddNTP has a different fluorescent tag attached. - All four reactions can be carried out in a single capillary. - This is much quicker than the previous methods. - Fluorescent peaks are detected each time the ddNTP is incorporated into the sequence, these peaks are recorded by a computer. - This produces a sequence trace.

- Sanger sequencing is used to detect mutations in DNA.

The 454 method - Have double stranded DNA that is broken into smaller pieces. - Adaptors are added to the DNA fragments, these help with the amplification of the fragments as well as sequencing. - Adaptors are short sequences of DNA. - Beads are then added to the mix and primer sequences on the beads are complimentary to the parts of the adaptors that have been previously added. - This allows the DNA fragments to bind to the primer. - The aim is to have only one DNA fragment bound to each bead at the beginning. - Amplification: the DNA fragments are copied numerous times on each bead. The beads are then filtered, removing any beads without DNA attached. - The DNA fragments are then denatured to produce a single strand of DNA. - The beads are put onto a well plate along with enzyme beads which contain DNA polymerase. - The enzyme beads attach to the DNA.

- Nucleotide bases are then fed in waves, and light is given of when each base is incorporated. - The intensity of the light given off is equivalent to the number of the same type of nucleotide that has been incorporated. - The method anchors DNA to resin beads, allowing DNA the sequence to be determined much faster than capillary sequencing. The Illumina method - Is a big commercial venture. - The starting DNA is fragmented to produce sequences of around 200-300bp in length. We are able to do this because we know the template of the genome. - Adaptors allow us to know there the DNA molecule is on the glass slide. - Adaptors are attached to each side of a DNA fragment that is around 200 to 300bp in length. - Adaptors are added to pin down the DNA fragments, so copies of the fragment can then be built up on the slide.

- Slides are used which have primers attached. The primers used are oligonucleotides. - The DNA fragments hydrogen bond to primers on the slide. - Each position on the slide can attach a different DNA fragment. - The adaptor molecules that have been added to the DNA fragments attaches to the primer through complimentary base pairing. - The DNA fragments need to be amplified on the slide in order to work with the sequences. - The aim is to produce multiple copies of the DNA fragments, and the experiment produces clusters of identical DNA. - DNA molecules are in constant motion; they have dynamics and are not rigid structures. DNA fragments are able to bend over and hydrogen bond with complimentary primers on the slide, through the adaptors on the end of the fragments. - DNA polymerase is added to the slide and the DNA fragments are copied. Remember the primers are already present on the plate. The strands are separated to provide a template for the DNA polymerase. - DNA replication is repeated a number of times – like PCR on a slide. - This produces a DNA cluster. - The DNA template is then ready to be sequenced. The primer used is complimentary to the original adaptor molecule. - This method can simultaneously copy all DNA molecules. - Fluorescent dNTPs are added to the slide and each time a complimentary base is incorporated a laser is activates fluorescence and the base is detected and recorded. - The computer is able to register each fluorescent even in every DNA cluster on the slide. - The computer then deciphers the events and provides a sequence for each fragment. Each incorporated base has a characteristic signal. This is a very sophisticated detection system. - A sequence is produced for each fragment. - ADVANTAGES: The method is cheap quick, and can be used to sequence entire genomes (Metagenomics). - DISADVANTAGES: The fragm...