biotechnology recombinant DNA and genomic analysis (1)

Researchers use recombinant DNA technology to carry out five basic operations Lecture Recombinant DNA and allied methods Fragmenting complex genome into pieces for analysis Restriction enzymes   Restriction enzymes fragment the genome at specific sites Restriction enzymes   The number of base pairs a restriction enzyme recognizes determines the average distance between sites and the size of fragments produced  Probability that a fourbase recognition site will be found in the genome = ¼ x ¼ x ¼ x ¼ = 1/256  cut DNA molecules at specific locations produce either blunt or sticky ends (cohesive ends) Fig 9.2 Fig 9.1 Different REs produce fragments of different lengths Cut long strings of DNA into fragments with restriction enzymes and separate them by gel electrophoresis Isolate, amplify, and purify fragments through molecular cloning Use purified DNA probes to identify similar sequences in libraries of clones or mixtures of DNA or RNA molecules Rapidly isolate and amplify previously defined genomic or mRNA sequences through the polymerase chain reaction (PCR) Determine the precise sequence of bases within isolated DNA fragments Time of exposure to a RE helps determine fragment size  The genetics use RE to produce DNA fragments of a particular length:   Complete digest: cutting at every recognition site Partial digest: cutting by controlling amount of enzyme and time the DNA is exposed to the RE when researches need the DNA fragments with expected lengths Fig 9.3 Partial digests are used so enzymes cut only a subset of the total number of recognition sites in a genome Gel electrophoresis separates DNA fragments according to size Preparing an agarose gel for electrophoresis Fig 9.4 Gel electrophoresis separates DNA fragments according to size Fig 9.5a Gel electrophoresis separates DNA fragments according to size Loading DNA fragments onto an agarose gel and performing electrophoresis Visualizing the DNA fragments on the agarose gel Fig 9.5a Fig 9.5a Visualizing the DNA fragments on the agarose gel Polyacrylamide gels separate very small fragments of DNA and agarose gels separate larger fragments Restriction maps can be inferred from DNA fragments cut with two enzymes providing a roadmap to DNA fragments and virus genomes  Steps to determine the order of restriction sites along a DNA fragment:      Divide a purified preparation of cloned DNA into three aliquots Cut one aliquot with EcoRI, one with BamHI, and the third with both enzymes Separate fragments by gel electrophoresis Determine the size by comparing to a size standard Use a process of elimination to derive the only arrangement that can account for the sizes of the fragments obtained in all three aliquots Fig 9.5b Restriction mapping Cloning fragments of DNA Genomes of animals, plants, and microorganisms are too large to analyze using gel electrophoresis and restriction mapping  Cloning is a means      to identify a specific DNA fragment within the genome, purify away all of the other fragments, and amplify it, making many identical copies of the fragment Such a fragment can then be analyzed by restriction mapping and DNA sequencing Fig 9.6 Two strategies to purify and amplify individual fragments Polymerase chain reaction  Purification and amplification of previously sequenced genomic regions  Molecular cloning  Purification and amplification of previously uncharacterized DNA 2.1 Step of molecular cloning      Ligation of fragments into cloned vectors creates recombinant DNA molecules Cut DNA and insert fragments of specific sizes into vectors Transport vector-insert molecules into living cells that make many copies DNA clones are any amplified set of purified DNA molecule Step of molecular cloning  Sticky ends facilitate recombinant DNA fabrication  Cutting the vector and DNA fragments generates complimentary sticky ends that increase the efficiency of ligation between the vector and insert DNA 2.2 Step of molecular cloning  Steps of molecular cloning Host cells take up and amplify vector-insert recombinants  Transformation – vectors carry insert DNA into cells     Creating recombinant DNA with vectors Recombinant DNA molecules are added to a suspension of competent E coli Cells are heat-shocked or shocked with a high-voltage electric shock (electroporation) Transformants are identified  Ampicillin resistance – cellular clones are colonies that represent each viable plasmid containing bacterial cell Plasmid-insert transformants are identified  b-galactosidase selection Cells that turn blue DO NOT have an insert b-gal gene is intact Cells that are white DO have an insert b-gal gene is interrupted by insert   Fig 9.7 b Steps of molecular cloning 2.3 Steps of molecular cloning: Purify cloned DNA Identification of transformed bacterial cells with plasmids and inserts Plasmids and inserts are separated from bacteria and vectors by centrifugation, restriction digests and gel electrophoresis Fig 9.9 a, b Fig 9.8 Video for DNA cloning  http://highered.mcgrawhill.com/olc/dl/120078/micro10.swf 2.4 Libraries are collections of cloned fragments  How to compile a genomic library  Complete genomic library – collection of clones that contains one copy of every sequence in the entire genome   Genomic equivalent – number of clones in a perfect library  Divide the length of the genome by the average size of inserts carried by the library’s vector Researchers usually make libraries with four to five genomic equivalents for a 95% probability that each locus is present at least once cDNA library construction cDNA libraries Whole genomic libraries contain all DNA in a genome cDNA libraries carry information from the RNA transcripts in a particular tissue  cDNA libraries contain only information from a gene’s exons     Prepare total mRNA from a tissue Add reverse transcriptase and four deoxyribose nucleotide triphosphates, and primers to initiate synthesis Fig 9.10 Expression vectors produce large amounts of specific polypeptide A comparison of genomic and cDNA libraries Vector contains promotor and regulatory sequences  Vectors transformed into bacteria, yeast, or cultured mammalian cells  Fig 9.11 Fig 9.12 a Screening an expression library  Hybridization is used to identify similar DNA sequences Expression library – entire cDNA library  Probe library with fluorescently labeled antibody that binds to protein product of gene     Fig 9.12 b Hybridization: DNA/DNA, DNA/RNA, RNA/RNA Prepare library  Distribute library’s clones on petri dish  Transfer clones to nitrocellulose disk Prepare probe  Previous cloned DNA  PCR fragment  Oligonucleotide Screen library  Expose probe to clones on nitrocellulose  Determine location of matching clone by autoradiography or fluorescence Fig 9.13 Hybridization in DNA microarray  DNA microarray Gel electrophoresis and hybridization to map DNA fragments  Southern blot      Fig 9.15 (cont‘) Fig 9.15 Polymerase chain reaction to rapidly isolate DNA fragments  Cut whole genomic DNA with restriction enzyme Separate DNA fragments by electrophoresis Blot fragmented DNA to a filter Hybridize to DNA probe Observe matched bands by autoradiography or fluorescence Oligonucleotide primers begin copying DNA PCR (polymerase chain reaction) achieved exponential accumulation of target DNA   Based on previously determined DNA sequence, develop short oligonucleotides (~ 20bp) complementary to sequences flanking the target DNA Oligonucleotides act as primers to copy DNA similar to DNA replication Each cycle of replication doubles amount of target DNA Exponential amplification PCR  Uses for PCR Genetic mapping Genotype detection  Analyze traces of partially degraded DNA  Evolutionary studies       Compare homologous sequences from related organisms Compare sequences from a variety of sources Studies of gene diversity PCR DNA Sequence Analysis All sequencing projects use same basic protocol Sequence determined approximately 800 bases at a time Maxim-Gilbert method  Chemical cleavage of DNA at specific nucleotide types  Sanger method  Enzymatic extension of DNA to defined terminating base  Sanger method most popular and efficient, particularly for automated methods  Both techniques approximately 99.9% accurate    Diagnosis of infectious diseases General Principals of Sanger Sequencing Method Fig 9.17 Fig 9.17 Automated DNA sequencing Fig 9.17 Fig 9.18 a Automated DNA Automated DNA Sequencing  Output from an automated DNA sequencing reaction  Each lane displays the sequence obtained from a separate DNA sample and primer Fig 9.18 c  Computer reads of the sequence complementary to the template strand from right to left (5’ – 3’ direction) Machine generates complementary strand Ambiguities are recorded as an “N” and can sometimes be resolved by a technician Fig 9.18 b Sequencing long regions of DNA Sequencing long regions of DNA  Primer walking    Sequence starting from both ends of cloned insert New primers derived from sequence obtained in previous round Shotgun sequencing  Long DNA sequence chopped into many small fragments which are cloned individually  Sequence of all small fragments determined  Small fragments aligned by computer to generate one long continuous sequence Fig 9.19 Rapid sequencing Shotgun approach relies on redundancy  Must gather sequence information on 3-4 times the actual number of base pairs from the original clone for full coverage  Sometimes must fill in gaps with primer walking  Must have many automated sequencers  Very fast if laboratory has enough equipment  Primer walking  No redundancy required  No alignment necessary  Slower than shotgun sequencing because must make primers after each round of sequence  Works well in laboratories without large number of automated sequencers  The genes encoding hemoglobin occur in two clusters on two separate chromosomes  The a-globin cluster contains three functional genes that spans 28 kb on chromosome 16 Understanding the genes for hemoglobin: a comprehensive example  Recombinant DNA technology used to isolate the a and b globin gene loci  Isolated RNA from red blood cell precursors  Produced cDNA libraries  Probed libraries for cDNA clones  Sequenced individual cDNA clones to identify a and b globin coding sequences  Used PCR to amplify sequences in many individuals with and without disease  Probed genomic DNA libraries to identify genomic clones and surrounding regions of globin genes  Used cDNA to probe Southern blots to determine number and location of coding sequences within genomic locus  Sequenced entire chromosomal regions containing a and b globin genes The genes encoding hemoglobin occur in two clusters on two separate chromosomes  The b-globin cluster contains five functional genes and two pseudogenes spanning 50 kb on chromosome 11 Fig 9.20 b Fig 9.20 a Locus control region (LCR) turns genes on and off The succession of genes in each cluster correlates with the sequence of expression during development   a – globin cluster    LCR associates with specialized DNA binding proteins at 5’ end of each gene cluster, bending chromosome back on itself to turn genes on and off in order  during the first five weeks of embryonic life Two a chains during fetal and adult life b – globin cluster     during first five weeks of embryonic life Two  chains during fetal life b and d within a few months of birth  Example where adult b-globin genes are removed   LCR cannot switch from activating fetal genes to activating adult genes Fetal genes remain active in adult Fig 9.20 c A variety of mutations account for the diverse symptoms of globin-related diseases  Hemolytic anemias Two general classes of disorders  Mutations alter amino acid sequence   Hemolytic anemias  e.g., sickle cell anemia – A-to-T substitution in sixth codon of b-globin chain Mutations that reduce or eliminate production of one or two globin polypeptides  thalassemia Fig 9.21(a.1) Sickle Cell Anemia Thalassemias Fig 9.21 (a.2) Fig 9.21 (b.1) 10 b-thalassemia Mutations in globin regulatory regions cause thalassemias Mutations in the TATA box can eliminate transcription causing b thalassemia  Fig 9.21 (b.2) Fig 9.22 a Mutations in globin regulatory regions cause thalassemias Evolution of the globin gene family   Mutation in the LCR can prevent expression of all a-globin genes causing severe a-thalassemia Fig 9.22 b Duplication of an ancestral gene followed by more duplications established the a and b-globin lineages Fig 9.23 11 ... to five genomic equivalents for a 95% probability that each locus is present at least once cDNA library construction cDNA libraries Whole genomic libraries contain all DNA in a genome cDNA libraries... fragments  Cut whole genomic DNA with restriction enzyme Separate DNA fragments by electrophoresis Blot fragmented DNA to a filter Hybridize to DNA probe Observe matched bands by autoradiography... for cDNA clones  Sequenced individual cDNA clones to identify a and b globin coding sequences  Used PCR to amplify sequences in many individuals with and without disease  Probed genomic DNA