Study guide exam 3 Chapters 10 11 12 16 17 Please also refer to lecture slides notes and homework questions Define recombinant DNA describe features of cloning vectors replication origin selectable marker cloning sites etc Give examples of different vectors and compare Recombinant DNA fragments of DNA inserted into a cut vector chromosome Cloning vectors fragments of donor DNA that are inserted into bacteria or their plasmids that carry and amplify the gene of interest Plasmid vector usually have drug resistant genes which provide easy distinguishing Bacteriophage can put double stranded DNA into phages which then get inserted into bacteria bacterial plasmid dna Fosmids can carry about 35 45 kb of DNA They are engineered hybrids of phage and BAC bacterial artificial chromosome most popular use for copying human genome they can carry up to 200 kb and its derived from the F plasmid cDNA how is it made reverse transcriptase Complementary DNA cDNA DNA transcribed from a messenger RNA template through the action of the enzyme reverse transcriptase The cDNA is made from mRNA with the use of a special enzyme called reverse transcriptase originally isolated from retroviruses To make cDNA a researcher purifies mRNA usually from a particular tissue such as pancreas or plant roots and adds it to a test tube containing reverse transcriptase the four dNTPs and a short primer of polymerized dTTP residues called an oligo dT primer The oligo dT primer anneals to the poly A tail of the mRNA molecule being copied Using this mRNA molecule as a template reverse transcriptase catalyzes the synthesis of a single stranded DNA molecule starting from the oligo dT primer DNA polymerase then copies the cDNA into a double stranded DNA molecule Define restriction enzyme digestion 3 5 overhang sticky ends and blunt ends Restriction enzymes cut long chromosomes into smaller sizes before they are inserted into vectors These enzymes cut at specific sites called restriction sites EcoRI cuts DNA and plasmids with palindrome sequences This forms sticky ends and allows DNA sequences to bind to bacterial plasmids Blunt ends PCR usually produces dna with blunt ends that can only be combined with ligase Instead PCR can be run at higher temperatures and palindrome sequences will be added to the dna Palindrome sequences recognized by restriction enzymes Palindrome sequences is a series of DNA in which both strands have the same nucleotide sequence antiparallel Usually code GAATTC CTTAAG These series are cut between the A and G on each strand and form overhang and sticky ends DNA Digestion with restriction enzymes and prediction of the results Process by which DNA is prepared for cloning and amplification in vectors Restriction enzymes cut DNA into segments at specific sites known as restriction sites Restriction sites appear by chance in DNA Here s a sample problem as seen in class Assume that a plasmid circular is 4000 base pairs in length and has restriction sites at the following locations 200 1000 1400 2600 Give the expected sizes of the restriction fragments following complete digestion A 400 800 1000 1000 B 400 800 1200 1600 C 300 700 2200 D 700 400 1400 2600 E 300 700 1000 1200 ANSWER B Read DNA bands on an electrophoresis gel compare and contrast Southern B blot Northern blot and western blot Southern Blot Puts DNA on gel but DNA must be denatured first Northern blot RNA is run on gel and doesn t require denaturing as it already in small enough fragments Western blot same as other blots but with denatured proteins Describe the chain termination Sanger method of DNA sequencing list the materials involved Read DNA sequence from a gel This sequencing technique uses dideoxy ribonucleotides in order to stop dna synthesis at different lengths Using DDNTP with N being ATC or G results in many different stands of Dna cut up according to where the DDNTP was inserted These fragments can be put on a gel and read to determine the sequence of DNA Draw and compare the structure of ribonucleotide deoxy and dideoxy ribonucleotide Ribonucleotide Deoxy Dideoxy no further nucleotides can be added Describe how PCR is done and compare it with DNA cloning describe what happens at different temperature during PCR Polymerase chain reaction in vitro a specific gene is isolated and amplified by DNA polymerase extracted from heat tolerant bacteria One round of PCR produces blunt ends but multiple rounds at higher temperatures produces sticky ends Genomic DNA and cDNA library construction and comparison Genomic DNA library is an entire genome copied into thousands of independent vectors The genomic library is necessary to make a clone cDNA library are all the exon gene encoding parts of the genome that are inserted into thousands of vectors cDNA library is useful for finding a specific gene that encodes for a protein Describe how gene knockout is done in mouse For gene knockout to occur the ES cells with the targeted mutation are added to the embryo of a mouse A vector is added to the cloned gene which is then inserted into the embryo The vector will be inserted into the targeted gene List ways in which gene expression can be regulated in prokaryotes and in eukaryotes What is the function regulatory elements Lac system Trp operon Gal 4 miRNA methylation Acetylation The lac system o I Repressor binds to operon in order to inhibit gene expression o Inducer lactose binds to repressor molecule and releases it from operon presence of inducer will cancel out repressor o Promoter where RNA polymerase binds to initiate transcription If a mutation is located on the promoter no transcription will occur o Operon Where repressor binds to stop gene expression A mutation on this will result in the repressor not being able to bind and gene expression will occur o Z Y A the genes that are being coded Mutations and what they mean o OC constitutive mutation cis acting repressor cant bind so gene expression occurs regardless of presence of inducer aka lactose o I recessive to I it means that the repressor has a mutation and cannot form Its constitutive and gene expression will occur regardless of lactose o IS repressor will bind to operon regardless of lactose These are dominant over I and result in no gene expression Trans acting o Z Y these mean that there is a mutation on the gene the codes for one beta galactosidase and permease These mutations indicate that the intended product cannot be formed on its strand Cis acting Lac catabolite repression if
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