Chapter 10 Study Guide.docx

Chapter 10 Study Guide Topic 1: Isolating and Amplifying specific DNA fragments (In vivo vs. In vitro) Key words to mention to the class so they don’t get lost during presentation:  Amplification: replication done to DNA segment we’re looking at o Can be performed with bacterial cells or in test tube (in vitro)  Donor DNA: the sample DNA we’re using, sometimes is the entire genome In Vivo: Parts of the Donor DNA are added into the plasmid or bacterial virus that will take over and amplify the gene we’re looking at. This will be called Vectors. 1. The Donor DNA are cut up using enzymes called restriction endonucleases as their molecular scissors 2. The scissors cut the DNA into smaller fragments 3. Each little fragment is added into vector chromosomes already cut to form recombinant DNA molecules 4. These recombinant DNA are transferred into bacterial cells - which is taken by each cell In Vitro: Is performed by PCR (Polymerase Chain Reaction):  It recognises the location of the gene by complementation on the short primes  Amplifies it by cycles to create many of them Both of the techniques require specification of a protein to notice the sequence of interest and also the formation of a ds molecule from a starting ssRNA/ssDNA. Topic 2: Generating recombinant DNA molecules Define Sources of Donor DNA (what we’re using the amplify and isolate DNA)  Genomic DNA: This is the entire genome. gDNA obtained directly from chromosomes of organism of interest. Before any cloning can be done, it must be cut up.  cDNA (complementary DNA): double stranded version of an mRNA molecule, is DNA copied by mRNA. Doesn’t need to be cut in order to be cloned. cDNA is created from mRNA with the use of reverse transcriptase, an enzyme that was isolated from retroviruses. The reason why it’s more useful to use cDNA in eukaryotes is because the introns have been spliced out in the genomic sequence.  Chemically synthesized DNA: a specific recombinant DNA that cannot be isolated. If the DNA sequence is known, then the gene can be chemically synthesized DNA is cut in small fragments of interest (restriction fragment) by using restriction enzymes that have a sticky end. Hybridization DNA palindrome (the strands are the same just in different directions) is cut by the restriction enzymes mostly stagger (offset). In EcoRI there are 6 nucleotides and when the palindrome is cut each strand has 5 nucleotides and one sticky ends. Under specific conditions, Donor DNA can be inserted by complementing the sticky end and by using DNA ligase to seal them. PCR In Vitro:

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Multiple copies of short primers binds to different ends of genes or regions to be amplified. Two primers bind to opposite DNA strands. Polymerases add bases to the primers and polymerization proceeds back and forth. Cycles of denaturation ( high and low), annealing and synthesis are repeated in the amplification process. DNA polymerase Taq polymerase, comes from the bacterium Thermus aquaticus, is extremely heat resistant. Kary Mullins made PCR possible and awarded Nobel Prize in Chemistry 1993

Cloning DNA fragments with blunt ends: “ connects harder because ddDNA”  I.e. cDNA and DNA fragments have blunt ends that come from PCR.  Not effective: blunt end fragments joined to the vector with only ligase.  Effective ways: 1. Create sticky ends of PCR products using specific PCR primers that contain endonuclease recognition sequences at 5’ ends. The last PCR product is digested with the restriction enzyme and produces a fragment to be inserted into a vector. 2. Add sticky ends to double stranded oligonucleotides called linkers or adapters that contain a restriction site. Linkers are joined by ligase to cDNA strands. Then to get sticky ends for cloning into a plasmid vector, DNA is incubated with the restriction enzyme. Amplification inside a bacterial cell: **  In bacterial cells, amplification takes advantage of prokaryotic genetic processes, including those of bacterial transformation, plasmid replication, and bacteriophages growth.  Cloning of Donor DNA Segments (Figure 10.8): -Recombinant vector enters bacteria wall → Colony of bacteria that contains single strand DNA inserts fused to its accessory chromosome (occurs after being amplified) → Replication of recombinant molecules exploit the normal mechanism used by bacteria to replicate chromosomal bacteria.  Steps for the amplification of donor DNA inside a bacteria cell: 1. Cloning vector introduces the gene of interest. 2. Recombinant DNA molecule goes inside host cell. 3. Amplification of the gene .  Generally the vector are small, able to replicate fast and contain one restriction site. Types of cloning vectors:  Plasmid vectors- are circular, small and independent.  Bacteriophage- a phage carries the DNA. (ex: Bacteriophage lambda carries 25-30kb)  Vectors that can contain larger DNA inserts (engineered): → Fosmids ( are made by a lambda phage and plasmid DNA, It carries 35-45 kb inserts and F plasmid) → BAC (Bacterial Artificial Chromosome) - F plasmid derivative (~7kb) can carry 100-200kb inserts. The Recombinant DNA is showed to a bacterium to cycle. Entry of recombinant molecules into the bacterial cell:  Foreign DNA molecules can go into bacterial cells through transformation (making the bacteria membrane competent), transducing phages ( the recombinants get a head and a tail. We mix it with the other phages so the gene can be passed) and infection (the

phage infects the bacterium and replicates, bacterium undergoes lysis and phage plaque is released).  To recover the recombinant DNA we destroy the vector and centrifuge it or we do electrophoresis. Genomic library: the resulting collection of recombinant DNA bearing bacteria or bacteriophages. Any recombinant clone represent just one mRNA, so in order to make sure that we have our segment of interest in the recombinant we need to cut the entire genome into fragments and amplify them. The total # of all these copies is called genomic library. The size of the genome is cut and distributed to the vectors. If vector carries only 20kb and the genome is 1000kb we are going to need 50 vectors to carry it. Also, to ensure that the segment of interest is going to be added to the recombinant DNA we multiply the amount of the clones by 5. Genomic library represents an average genome at least 5 times the amount presented. cDNA are collection of clones that contain just protein-coding regions and is obtained depending on the location where the gene is expressed. Topic 3 Finding a specific clone of interest Finding DNA After the cDNA are created we need to find our recombinant DNA of interest We can localize it by using probes, which are small derivative of relative cDNA (for complementation) or protein that is produced by the gene of interest (from protein to mRNA to gene of interest) Probes can recognise:  A nucleic acid  Specific protein → Autoradiogram Everything consists in complementation so the molecules are ss. The vectors are on a petri dish and a membrane containing probes (usually radioactive isotopes or fluorescent dyes) for complementation is above the vectors. The membrane is removed and put on a X-ray film to notice the location. So we go back to the petri dish and now we know which spots contain the gene of interest to make a colony of it in order to amplify it. Finding Protein We can find the gene by knowing the protein that it produces. All our clone vectors are in the petri dish and they are going to produce “fusion” proteins. We lay a membrane above it and we transfer the membrane containing the proteins in a petri dish with antibodies to recognise the gene that created the protein. We use “secondary antibodies” to localize the primary antibody by using X-ray film. Functional complementation (mutant rescue) We can restore a wt phenotype to a mutation sequence by using the wt gene to complement it. Make cDNA of a+ allele, insert the allele a+ into mutant sequence, notice in which clones the wt phenotype was restored and collect the bacteria that expressed it. Southern- and Northern- blot analysis of DNA

To analyse the DNA we use a technique called blotting (gel electrophoresis) by separating the part depending on their size (analyse). If the size is concentrated we can purify the DNA (preparative). Southern blotting (DNA imprinting) Northern blotting (RNA imprinting + location of expression and environmental conditions) Topic 4: Determining the Base Sequence of a DNA Segment In order to determine the nucleotides and its biochemistry in the gene of interest we perform the technique of Sanger sequencing (dideoxy sequencing). It consist in the ability to stop the DNA synthesis by the present of a modified nucleotide dideoxynucleotide (~ddNTP). In a primer DNA we add DNA pol, 4 dNTP (dATP, dTTP, dCTP and dGTP) and 1/chase ddNTP (ddATP/ddTTP/ddCTP/ddGTP). So depending on the ddNTP we are using we are going to stop the DNA synthesis at the dNTP, eg: ddCTP will stop when it added instead of dCTP. We are going to have 4 reading (1/N) that is going to reveal us the location in the DNA sequence. If we have an automatic sequencer we proceed the DNA sequence once and simultaneously the location of 4N will appear.