Unformatted text preview:

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 CellI) DNA Manipulation:a. Restriction Enzymes – cut DNA in well defined sequencesi. 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 Electrophoresis1. 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 gela. 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.ii. Introduce plasmid to bacteria.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 mRNAi. Take mRNA and hybridize oligo-dT primer to it, which binds to Poly-A tail on mRNA.ii. Use reverse transcriptase to make DNA copy, extending that oligo-dT tail. 1. Copied strand is cDNA strand.iii. Degrade RNA with RNase.iv. Synthesize Complementary DNA Strand1. Use DNA Polymerase to do this.2. RNA Fragment acts as primer.d. Polymerase Chain Reaction (PCR)i. DNA Denatures at high temperatures1. 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 sequence2. 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 Diagnosisi. Use of PCR to detect diseases in blastocysts.1. Allows one to select particular eggs that avoids disease.II) DNA Sequencinga. 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.b. In the past, genes were cloned and added to a genomic library.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.a. No OH group at the 3’ end of DNA, so nothing for next base to attach to.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 stopsit.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), wecan tell where on the gel that base pair ends up.2. Smallest base pairs will travel fastest down gel. c. Whole Genome Shotgun Methodi. 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 Sequencingi. 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. iii. Imaging based detection of fluorescent labels1. 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.3. Incorporate another colored base.4. Repeat.5. You end with several cycles, telling the sequence. III) RNA Analysisa. 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 RNAi. 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 Hybridization1. Use a probe to hybridize to mRNA wherever they are produced in cells.2. These probes bind between boundaries in exons, where mRNA is 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


View Full Document

BU BE 209 - Final Review

Download Final Review
Our administrator received your request to download this document. We will send you the file to your email shortly.
Loading Unlocking...
Login

Join to view Final Review and access 3M+ class-specific study document.

or
We will never post anything without your permission.
Don't have an account?
Sign Up

Join to view Final Review 2 2 and access 3M+ class-specific study document.

or

By creating an account you agree to our Privacy Policy and Terms Of Use

Already a member?