Final Exam

! Cumulative, but material after first midterm emphasized ! Two questions on Ghost in Your Genes ! 75 questions total: 2 pts each

Since last midterm

! Replication ! Repair
! Know common types of mutations and how they are repaired ! Know repair pathways and types of enzymes involved

! Recombinant DNA technology

! Restriction enzymes, cloning ! Genomic vs cDNA libraries ! Westerns, Sequencing, PCR

! HIV
! How it is tested for (ELISAs, westerns, RT-PCR) ! Life Cycle ! What steps drugs block

Recombinant DNA tech. cont

! Western blots ! PCR ! DNA sequencing

Detection of proteins using Western blots

! Separate proteins by size using SDS gel electrophoresis
! SDS denatures protein and coats it with negative charge ! Smallest proteins migrate faster in gel
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! Transfer to membrane ! Detect specific proteins with antibodies ! Detect protein-antibody using enzyme conjugated secondary antibodies

Second antibody recognizes first antibody and is linked to either an enzyme (horse radish peroxidase (HRP)) or a fluor

An alternative to cloning (or used in conjunction with it)

! PCR: Polymerase chain reaction ! Method to amplify specific regions of the genome in the test tube ! Based on in vitro DNA synthesis ! Has many different uses revolutionized molecular biology

PCR

Specific amplification of gene X from genomic DNA

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PCR Reaction
! PRIMERS: Oligonucleotide primers which bound the region to be amplified are made ! POLYMERASE: Taq polymerase, isolated from Thermus aquaticus, is resistant to heat denaturation ! SUBSTRATES: dNTPs, buffer, Mg2+ ! REACTION: Repeated many times to amplify a specific region from as little as one copy of DNA

Cycle 1

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synthesizes DNA

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Cycle 2

1: 94oC, DNA denatured 2: 42oC, primers hybridize 3: 72oC, DNA synthesized

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The process is repeated 25-35 times, to yield over 1 billion copies of the amplified region from a single copy of DNA

PCR has changed the way molecular biology is done

! Allows one to amplify DNA before cloning it (or instead of cloning it) ! Allows one to check recombinant constructs using PCR instead of isolating DNA and cutting with restriction enzymes ! Allows one to screen for diseases or verify DNA from suspects using very small samples

qPCR allows one to accurately quantitate the amount of DNA you are amplifying using a fluorescent dye

DNA Sequencing
Maxam-Gilbert Sequencing Based on chemical degradation of DNA (We wont be covering this)" Sanger Sequencing Based on premature termination of DNA synthesis using dideoxynucleotides

Sanger or Dideoxy Sequencing

1. DNA to be sequenced is first denatured 2. Oligonucleotide of known sequence is hybridized to template DNA"

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DNA to be sequenced
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Oligonucleotide as primer

3. Template-primer is distributed in four different tubes 4. To each tube are added all the substrates for DNA synthesis plus one dideoxynucleoside triphosphate

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5. In vitro DNA synthesis produces a collection of fragments, each terminating with the dideoxynucleoside (in this case ddG)

6. Fragments are denatured and separated on polyacrylamide gel. Bands are visualized by autoradiography !"#

Autoradiogram of Dideoxy Sequencing Gel

Automated sequencing uses fluorescent labels instead of radioactive labels. ddA-green tag ddG-black tag ddT-red tag ddC-blue tag
1.! One reaction containing all four dNTPs and all four labeled ddNTPs. 2.! PCR used to amplify sequencing products (only 1 primer) 3.! Fragments separated by electrophoresis in a single lane. 4.! A fluorescent detector records the color of the passing bands and generates a sequence.

HiSeq is the latest in new automated sequencing Whole genomes can be sequenced for less than $1000