L53-07 DNA tech SLIDES - L. 53 DNA Technology Studying how...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: L. 53 DNA Technology Studying how gene functions often requires: single gene away from other genes in the genome - solution: restriction enzymes can cut DNA at specific sequence methods to produce large quantity of the gene so that its function can be observed easily - solution: vector such as plasmids in bacteria can produce many copies of the same gene Restriction Enzyme - Restriction enzymes are specific enzymes in bacteria that cut foreign DNA for defense - After being "restricted" the foreign DNA can be digested by exonucleases, enzymes that digest DNA from ends - In contrast, restriction enzymes are also called endonucleases endonucleases exonucleases Restriction Enzyme Mechanism - Restriction enzymes recognize specific sequences (restriction sites) of DNA and cut both strands in specific ways - The sequences recognized are 4 to 8 base pairs - The sequences are palindromes - The average frequency of restriction sites is 1/44 (1/256) to 1/48 (1/65536) - Restriction reaction creates reproducible DNA segments called restriction fragments Restriction Enzyme Cutting - Some RE cut create blunt ends - Some RE cut create staggered ends Blunt cutter Stagger cutters Fig. 20.3 - The staggered ends are often called sticky end for their tendency to re-associate with each other EcoRI Molecules cut by the same restriction enzyme can form base pairing Gap in sugar-phosphate backbone is sealed by adding ligase Restriction Enzyme Structure - Most restriction enzymes are homodimers BamH1 GGATTC CTTAGG Protection From Own Restriction Enzymes GAATTC CTTAAG EcoRI methylase CH3 - Restriction sites are also in bacteria's genome - Protection from being cleaved by own restriction enzymes is through methylation at the restriction sites GAATTC CTTAAG EcoRI (restriction enzyme) CH3 No cleavage Restriction sites DNA 1 2 3 2 3 1 Electrophoresis - Gel made of agarose or polyacrylamide forms a matrix - Restriction fragments loaded into gel well at the negatively charged end - Apply electrical current - Fragment of different sizes are separated Fig. 20.8 A 1 2 3 mutation B 1 2+3 S large A B 2+3 2 3 1 small Southern Blot - Uses DNA probe to detect DNA fragments separated by gel electrophoresis - Restriction fragments undergo electrophoresis - Special paper pressed against the gel - Gel and paper put on alkaline solution nitrocellulose paper Southern Blot Fig. 20.10 Southern Blot Cloning - Cloning of individuals - Cloning of cells - Cloning of genes or DNA fragments Cloning of genes or DNA fragments Components involved: gene of interest plasmid vector bacterial cells Phage vector Plasmids - Extra chromosomal, circular, double-stranded, haploid DNA - Naturally occur in bacteria, yeast and other eukaryotes - Live as a symbiont or parasite in their hosts - Some replicate independently from host's chromosomal DNA Plasmids as Vector In molecular biology, vectors are carrier of gene of interest Plasmid is the most common vector Plasmid used as vector must possess three components 1. Origin of replication: to replicate independently from host 2. Multiple cloning sites (polylinker region): restriction sites for genes to be inserted in 3. Selective marker: gene whose product allows host cell to grow in the presence of inhibitor Plasmid Vector Cloning Gene or DNA Techniques involved: Recombination: combining gene or DNA of interest with the cloning vector (such as plasmid). The outcome is recombinant DNA Transformation: permanently change the genetic makeup through acquiring exogenous DNA, e.g., bacterial cells pick up plasmids from the environment - To distinguish transformed and untransformed cells Selection: using the selective marker on plasmids Human gene EcoRI ends Ampicillin resistance gene Fragment joined with DNA ligase Recombinant plasmid E. Coli cell transformed with recombinant plasmid Some cells are transformed, some not Cells plated onto medium containing ampicillin Only cells containing recombinant plasmid survive to produce resistant colony Fig. 20.4 Genomic Library - Entire collection of a species' genome stored in plasmids or phage Selecting a particular clone in a library-screening Library is formed in a mixture of all DNA fragments To identify one clone from others requires extra techniques - Screening library: methods to identify and isolate a particular clone - Use a probe (either DNA or RNA) to hybridize the clones ? Screening Library Using Probe special filter colonies containing vectors of interest expose filter to photo film lyse bacteria and denature DNA with alkali - A single plate can produce many filter samples - Original colony will be isolated and cloned Polymerase Chain Reaction selects a portion of genome to make copies Marker region DNA Polymerase Chain Reaction (PCR) PCR is a method of amplification of DNA sequence of interest rapidly An in vitro reaction (cell-free) Required materials - template: the DNA sequence to be copied - dNTPs (dATP, dGTP, dCTP, and dTTP) as building blocks - primers: short DNA sequences complementary to the template to start the reaction - DNA polymerase: to make reaction happen Primers are needed to start DNA replication Primers - Short nucleotide sequences complementary to DNA of interest - One pair of primer is needed for each DNA marker Forward primer Reverse primer DNA Temperature Profile for PCR 94C 50C 72C Outcome Number of copies = m 2n m= initial number of template DNA n = number of thermal cycles Applications - Diagnosis for genetic disease - Phylogenetic studies - DNA from ancient mummies or fossils - Crime scene samples - Many more! http://lasp.colorado.edu/education/yellow_stone/Images/skyimage_large.jpg DNA polymerase extracted from bacteria living in hot spring in Yellowstone National Park Sanger or Dideoxy Sequencing Based on premature termination of DNA synthesis using dideoxynucleotides Required ingredients Sequence of interest as template A primer DNA polymerase dNTPs Dideoxyribonucleoside triphosphate (ddNTP) ddNTP lacks 3'-hydroxyl group Incorporation of ddNTP terminates polymerization -Each of the four ddNTP is labeled with a different fluorescent color - Reaction tube has all ingredients including low quantity of ddNTP - Fragments terminated randomly during elongation, covering all possible lengths - Color of each fragment indicates base at which termination occurs Fluorescent sequence printout DNA Sequencing Frederick Sanger -1980 Nobel prize winner -1958 Nobel Prize winner for protein structure ...
View Full Document

This note was uploaded on 05/18/2008 for the course BIO G 104 taught by Professor Chen,k.c during the Spring '06 term at Cornell University (Engineering School).

Ask a homework question - tutors are online