Molecular Biology BE 209 Final Review by Matthew Brown Biomedical Engineer Major Test will be 90 this material 10 previously tested material This guide focuses on the 90 Review previously administered exams and acceptable answers for them to understand the remaining 10 or watch old review videos DNA Manipulation Stem Cell I DNA Manipulation a Restriction Enzymes cut DNA in well defined sequences i Cleave at specific sequences ii Can leave either sticky or blunt ends 1 Sticky Ends can be used to attach other DNA strands iii These can cut at four six or eight bases 1 Enzymes that recognize four cleaves more than enzymes that recognize eight iv The cut DNA is separated using Gel Electrophoresis 1 Use of DNA s negative charge and positive and negative poles 2 Smaller DNA migrates through gel faster toward positive pole 3 Ethidium Bromide is added for fluorescence so we can see it v Identify specific DNA with Southern Blots 1 Nitrocellulose paper added to gel a Capillary Action causes DNA to move i Attaches to nitrocellulose paper 2 Paper thrown in plastic bag with radioactive probe a Hybridizes to specific sequence i Allows for visual of specific type of DNA b Ligase can bond cut DNA using ATP i Allows us to take PCR products and add it to a digested plasmid that was also cut with a restricted enzyme due to sticky ends 1 Plasmid usually has genes for antibiotic resistance and other proteins Introduce plasmid to bacteria ii iii Put those bacteria in cell culture with antibiotic in it iv We receive replicated bacteria with our plasmid v Can overexpress mRNA to overexpress protein c Complementary DNA cDNA from mRNA i Take mRNA and hybridize oligo dT primer to it which binds to Poly A ii Use reverse transcriptase to make DNA copy extending that oligo dT tail on mRNA tail 1 Copied strand is cDNA strand iii Degrade RNA with RNase iv Synthesize Complementary DNA Strand 1 Use DNA Polymerase to do this 2 RNA Fragment acts as primer d Polymerase Chain Reaction PCR i DNA Denatures at high temperatures 1 First step is to denature DNA to separate strands 2 Attach primers to original strands by lowering temperature a Excess primers will attach quickly 3 Extension by DNA synthesis ii This process is repeated and DNA is amplified iii You can amplify cDNA with RTPCR Reverse Transcriptase PCR iv In order to pick primers 1 Look for 20 23 bases before start sequence and after end sequence 2 That series of bases ends in CC CG or GG 3 At the end primer sequence use the 5 3 complement of the sequence you see and flip it a Both primers should be reading from 5 to 3 left to right e Pre implantation Diagnosis i Use of PCR to detect diseases in blastocysts 1 Allows one to select particular eggs that avoids disease II DNA Sequencing a Source DNA fragmented cloned with bacteria technique previously discussed i But what do we do after we have the cloned plasmids 1 Have to sequence them In the past genes were cloned and added to a genomic library b c Shotgun Sequencing i taking random clones of DNA and sequencing them based on overlap d Sanger Method i Chain Termination Method 1 Use of dideoxynucleotide triphosphates ddNTPs as DNA chain terminators base to attach to a No OH group at the 3 end of DNA so nothing for next b normal amount of deoxynribonucleoside triphosphates dNTP with hydroxyl groups on their 3 ends small amount of one ddNTP an A T C or G i sequencing happens normally and then ddNTP stops it ii Happens at a random place so there are multiple strands of different sizes iii Denature and separate those strands iv The DNA strands can be ran on a gel 1 By labeling the ddNTP with colors representative of the bases A T C or G we can tell where on the gel that base pair ends up 2 Smallest base pairs will travel fastest down gel c Whole Genome Shotgun Method i the genome is broken into smaller overlapping fragments ii each fragment is sequenced iii genome assembles based on overlapping sequences iv Basically two fragments are created an overlap is found and the fragments combine with the overlap merging the two v Problem is that there are many areas of repetitive DNA 1 Shotgun sequencing may cause loss of info a The solution was to sequence from both ends e 2nd Generation DNA Sequencing i DNA is fragmented and primers are ligated on ii PCR done to create a huge array of shotgun fragments Not too close or overlapping on the surface Imaging based detection of fluorescent labels iii 1 Done through cyclic iteration a Adding different colored bases at different steps i Like a horizontal cut along the vertical strands of fragment which will show a particular base at a particular time ii This cyclic analysis continues until the strands are fully analyzed iv DNA on the surface can only be amplified if it bends over and hybridizes with a primer also on the surface 1 All duplicated strands would be very close to each other v Once the amplification has been completed incorporate differently colored nucleotides to the surface 1 Once that is completed you can image the strands to see exactly which got incorporated 2 Get rid of the fluorescent part afterwards and remove the protecting group on 3 hydroxyl Incorporate another colored base 3 4 Repeat 5 You end with several cycles telling the sequence III RNA Analysis a All cells have an identical genome with exceptions i Yet due to the expression of genes differences arise b Microarrays quantify expression of all genes i Measurements only give amount not activity c Methods of Analyzing RNA i Northern Blots 1 Like Southern Blots but loading RNA into a gel and separating with electrophoresis 2 Nitrocellulose paper is added yet again and a weight is added to keep it pushed down onto gel RNA moves up to nitrocellulose paper 3 Placed in a sealable plastic bag and single stranded probe DNA is added 4 Probe hybridizes with RNA of interest 5 By running the RNA in lanes that represent different tissues you can see the expression of RNA in that particular tissue ii In Situ Hybridization 1 Use a probe to hybridize to mRNA wherever they are produced in 2 These probes bind between boundaries in exons where mRNA is cells in large amounts 3 The probes have fluorescent molecules are enzymes that produce colors wherever the probe sequence is retained 4 Darker colors mean larger expression iii RT PCR Reverse Transcriptase PCR Isolate and purify mRNA 1 2 Reverse Transcription 3 PCR Amplification 4 Run on a Gel 5 Remember the process oligo dT bonds to Poly A Tail Reverse Transcriptase