General Biology Notes 11 5 13 11 05 2013 How are scientists able to determine the details of expression and regulation of genes DNA technology o Facilitates sequencing and manipulation of Organisms DNA and analysis of gene expression o DNA sequencing uses principles of base pairing to determine gene s complete sequence of nucleotides DNA cut into fragments and each fragment is sequenced by automated sequencing machines First procedure developed uses dideoxyribonucleotide chain termination technique dideoxy Dideoxy lacks the hydroxyl group at the 3 carbon If no hydroxyl exists at the 3 carbon then there can be Dideoxy nucleotides are abbreviated ddGTP ddCTP no chain elongation ddTTP ddATP Frederick Sanger method of DNA sequencing Can sequence fragments between 800 and 100 base pairs bp in length Fragment of DNA to sequence is denatured into single strands incubated in test tube with Primer DNA polymerase The 4 DNA nucleotides dGTP dATP dTTP CTP The four dideoxynucleotides which are labeled with a fluorescent tag ddATP ddGTP ddCTP ddTTP Figure 20 3 in book Synthesis of each new strand starts at 3 end of the primer continues until dideoxyribonucleotide inserted done at random Cant add anything after dideoxyribonucleotide Set of labeled strands of various lengths generated Color represents last nucleotide in each sequence Next generation sequencing Most recent development in DNA sequencing Doesn t rely on chain termination DNA fragments 400 1000 bp are amplified copied yielding large of identical fragments Sequencing of the fragments can be done in parallel Allows for 100 s of millions of nucleotides to be sequenced simultaneously within hours Specific strand of each fragment immobilized and complementary strand synthesized one nucleotide at a time monitored in real time sequencing by Figure 20 4 in book Fragmentize DNA Put different size strands into each solution with a synthesis bead Then we go through PCR to make 100s of thousands of copies It also denatures the double strand into a End with a bead that has the copied fragments single strand attached to it Then we add primer and DNA polymerase and put the millions of fragments into wells Sequentially add A C T G each washed away after they were done individually done to see which will stick at certain times this gives us the sequence of nucleotides Sequencing can be used to explore fundamental questions about evolutionary relationship and how life works In 2003 the human genome sequence was completed several years and 1 bill In past 10 years we have sequenced 4k bacterial 190 archael and 180 eukaryotic 17000 other species are being sequence at the moment Genome sequences for cells from several cancers ancient humans and bacteria in our gut It is projected that in near future we will sequence human genome in 6h on 900 machine o DNA cloning copies Allows scientist to work with single genes by preparing well defined segments of DNA in multiple identical These identical copies are very often made using E coli E coli are soo good for DNA coding because Have a large circular DNA molecule A smaller circular DNA molecule plasmid contains few genes not required for survival under most conditions To clone pieces of DNA using bacteria plasmids are isolated and foreign DNA inserted Known as a recombinant plasmid Bacteria is now known as recombinant bacteria Figure 20 5 E coli goes about replicating this plasmid with the inserted DNA sequence thus making fragments that could be taken out of the plasmid Dividing recombinant bacteria pass genes to descendants The rapid replication rate of bacteria make them ideal for DNA cloning Production of multiple copies of a single gene is a type of DNA cloning called gene cloning The plasmid inserted acts as a cloning vector A DNA molecule that can carry foreign DNA into a cell replicate there Plasmids are often used because Readily obtainable commercially Can be manipulated to insert foreign DNA in vitro Can be easily introduced into bacterial cells Gene cloning has 2 basic purposes 1 To amplify a particular gene to make copies 2 To produce a protein or product Figure 20 5 in book Enzymes called restriction endonucleases restriction enzymes are used to make recombinant DNA plasmids Restriction enzymes cut DNA molecule at a limited number of specific locations They are naturally produce by bacteria for Defending against phages viruses that infect bacteria Cutting up foreign DNA trying to incorporate into a bacterium s genome Hundred have been identified and isolated Each very specific recognize particular short DNA sequences restriction sites Enzymes cut both strands of DNA within the DNA of bacteria cell is protected from own restriction site restriction enzymes A s or C s within site normally recognized by enzyme are methylated add CH3 Most restriction sites are symmetrical palindromes Sequence of nucleotide in palindrome is the same on both strands when read in the 5 to What the restriction enzyme looks for and Can vary in length and actual nucleotides but enzyme is made to recognize certain 3 direction recognizes sites Enzyme cuts double stranded nucleotide by cutting the covalent bonds that hold the backbone of the strands together Exposes sticky ends o Regions of bases that are not hydrogen bonded Restriction enzymes and DNA ligase are used to make a recombinant DNA plasmid Figure 20 6 Use same restriction enzyme to cut both the inserted DNA and the plasmid This means the sticky ends will be similar and complimentary DNA ligase seals the inserted DNA into the plasmids be recreating the covalent bonds between the backbone of the plasmid and the newly inserted DNA Can take the recombinant plasmid and re insert it into the bacteria To check recombinant plasmid after it copied many times in host cells can cut products with same restriction enzyme Figure 20 7 Can expect yield of 2 kinds of DNA fragments 1 size of original plasmid 1 that s the size of the inserted fragment of DNA Gel electrophoresis used to separate and visualize DNA fragments of different lengths o A gel agarose is used as molecular sieve to separate nucleic acids or proteins by size electrical chard and other physical properties such as methylation of bases Molecules are sorted into bands by their size DNA can also be amplified in vitro using Polymerase Chain Reaction PCR Any given gene or fragment of DNA may represent a millionth of total DNA in cell so it is important to amplify when studying genes fragment cycler PCR is method for