MCB 2050 Midterm

What are the characteristics of the pBluescript cloning vector?

-bacterial origin of replication (to allow plasmid replication) -multiple cloning site (MCS) to allow insertion of foreign DNA -antibiotic resistance gene for selection of bacteria transformed with the plasmid -selectable marker (lacZ) to differentiate cells with plasmids containing inserted foreign DNA from those without (blue/white screening) -lac promoter to express mRNA (small (3kb) to allow for large insertions) -all RE sites in the MCS are unique sites in the plasmid

What are the main techniques for DNA and RNA analysis?

-electrophoresis -restriction mapping -Southern and Northern Blotting -PCR -DNA sequencing

Describe the elements/steps of the transfer apparatus for Western blotting

-protein gel sandwiched next to a blotting membrane -negatively charged proteins migrate out of gel towards positive electrode and are trapped onto the blotting membrane -membrane is incubated with a blocking solution (usually 5% milk) to prevent non-specific binding of antibodies to the membrane

Give the four (very general) steps of positional cloning of disease associated genes

1) Genetic mapping 2) Physical mapping 3) Transcript mapping 4a) Gene sequencing 4b) Transformation of mutant organism with wild-type gene

1) What does it mean to generate a DNA 'library'?

1) to clone several different DNA restriction fragments with cohesive ends complementary to the one in the MCS in the plasmid + many more recombinant plasmids with different EcoRI fragments of mouse DNA

Bacteria containing the Bluescript plasmid that do not have foreign DNA inserted in the MCS of the plasmid will be what colour when grown on agar plates containing X-gal?

Blue

What does CODIS stand for and how do the FBI use it? Example?

Combined DNA Index System: look at 13 different STR loci; for each 'allele' there is a different number of repeats of the same STR -eg: on chromosome 5, one allele might have 10TAGA repeats and the other 20 TAGA repeats

How does DNA ligase create a recombinant DNA molecule?

DNA ligase joins(ligates) the complementary single stranded ends produced by restriction enzyme cleavage

What are the two ways in which you can screen DNA libraries for genes of interest?

Genetic Selection and Molecular Hybridization

The name of the part of a cloning vector that has several restriction enzyme sites is called?

Multiple Cloning Site (MCS)

The technique used to identify a specific RNA following electrophoresis in an agarose gel is called?

Northern blotting

Compare the reporter plasmid and expression plasmid (2 for expression, three for reporter)

Reporter Plasmid: -the reporter gene does not have a promoter to control its expression -MCS: insert regulatory gene sequences here -the expression level of the reporter gene will tell you the efficiency of the regulatory sequences that you inserted Expression Plasmid: -promoter to control expression of your gene of interest -MCS: insert gene of interest (the gene you want to express) here

The technique used to identify a specific DNA fragment following electrophoresis in an agarose gel is called?

Southern blotting

After the third PCR cycle, how can you tell if newly synthesized DNA is a target copy or not?

The ends should be blunt with no overhang

1) How do you get from the RFLP to the gene of interest if you don't have a contig map? Give the steps

You 'walk' there 1) prepare a restriction map of RFLP clone that is close to gene of interest based upon pedigree data 2) subclone the fragment at the end of the RFLP 3) radioactively label this fragment and use it to screen (colony hybridization) genomic library 4) fragment farthest from the original probe is used to rescreen the genomic library 5) analyze each clone for presence of an ORF and continue to walk the chromosome until gene of interest is found -with each chromosome walk you test the new DNA fragment as a candidate for the disease gene; obtaining the sequence of the wild type and mutant allele will help confirm that the disease gene has been found

A collection of bacteria in which each colony contains a plasmid with a unique fragment of genomic DNA is called:

a Genomic library

What technique would you use to: a) determine whether a foreign DNA was inserted into a plasmid in the correct orientation b) determine whether a foreign DNA sequence has been integrated into the genome of a transgenic organism c) determine whether the levels of a gene transcript are altered during development d) measure the abundance of a specific protein in a cell extract e) visually determine the cellular localization of a protein

a) restriction mapping b) southern blot or PCR c) northern blot or RT-PCR d) Western blotting e) immunofluorescence or GFP-tagging

In the Bluescript cloning plasmid, the MCS is located within the reading frame of what gene?

beta-galactosidase (lacZ)

What kind of ends do the following produce: Sma1 Kpn1 BamH1

blunt 3'overhang 5'overhang

A library made from cellular mRNA is called:

cDNA library

The kind of ends following restriction endonuclease digestion that results in single stranded DNA overhangs are called:

cohesive (or sticky) ends

The kind of plasmid you would use to produce a foreign protein in cells containing the plasmid is called?

expression vector

The process by which a specific DNA or RNA on a membrane is detected using a radioactively labelled DNA probe is called?

hybridization

What is Southern (for DNA)/Northern (for RNA) blotting used for?

identification of homologous sequences in a gel blot between DNA/RNA target in a blot and the DNA 'probe'

What is restriction mapping used for?

physical mapping of DNA based on relative location of RE sites

What is the generic name of enzymes that recognize palindromic sequences in dsDNA and cleave the DNA at that location?

restriction endonucleases

What is electrophoresis?

the use of an electric field to separate macromolecules in agarose (DNA/RNA) or polyacrylamide (DNA or protein) gels

How does the green fluorescent protein act as a protein tag?

to help study subcellular localization of proteins by cloning GFP "in frame" with the open reading frame of your gene and view in real time by UV fluorescence microscopy -the tag can be placed at the N or C terminus of your protein

Overall, what is the Sanger dideoxy chain termination method?

a DNA synthesis reaction with chain elongation being terminated randomly by the incorporation of a fluorescent ddNTP (ddATP, ddCTP, ddGTP, ddTTP)

What is a clone library?

a collection of DNA enzyme restriction fragments cloned into a plasmid, each separated into different bacteria, each isolated on an agar plate to produce colonies

What are the applications of DNA restriction mapping?

-the relative positions of restriction sites on a DNA are used as markers; it provides an overview of the organization of a DNA fragment, eg mapping the boundary of a gene or checking the presence of a DNA insert -the restriction sites are also used as genetic markers for diagnostics--RFLP (restriction fragment length polymorphism) -can be used for cloning sub-fragments of the DNA and assembling a total genomic DNA sequence

1) What does Giemsa staining show? 2) What is the process of Giemsa staining?

1) 'G' bands in the human karyotype 2) mitotic chromosome smear, treat with the protease trypsin, stain with Giemsa, observe by light microscopy, photograph and arrange chromosomes

Briefly give the steps of the construction of DNA libraries

1) Cloning vector is cleaved with restriction endonuclease 2) DNA fragment of interest is obtained by cleaving chromosome with a restriction endonuclease 3) fragments are ligated to the prepared cloning vector 4) DNA is introduced into the host cell 5) Propagation (cloning) produces many copies of recombinant DNA

1) Do promoters have directionality? 2) What must eukaryotic expression plasmids contain?

1) Yes 2) a promoter upstream of the MCS and a polyA signal downstream of the MCS

Give the three steps for the construction of cDNA libraries

1) cDNA is synthesized by reverse transcriptase using oligo dT primer 2) mRNA is degraded by ribonuclease H 3) second strand cDNA synthesis using a DNA-dependent polymerase

1) What is the bluescript blue/white screening strategy called? 2) How is the E.coli lacZ gene involved in this? 3) Describe what happens to a colony containing a plasmid without the insert. 4) Describe what happens to a colony containing a plasmid with the insert.

1) insertional inactivation 2) it encodes beta-galactosidase which convert the colourless substrate Xgal into a blue product 3)- functional lacZ gene -functional beta-galactosidase enzymes convert X-gal (colourless) to a blue coloured product -colonies are blue 4) -non functional lacZ gene -functional beta-galactosidase enzyme not made -colonies are white

1) How is cDNA synthesized from mRNA? 2) What is ribonuclease H used for? What come next? 3) What kind of polymerase is used for the second strand? 4) What happens to the dscDNA fragments?

1) -by using a primer (usually oligo-dT since mRNAs all contain a 3' poly-A tail) and the RNA-dependent DNA polymerase reverse transcriptase (synthesizes DNA from an RNA template) 2) to remove RNA; second strand synthesis 3) DNA dependent DNA polymerase 4) they are inserted into cut plasmids

1) What are the three steps of a PCR reaction, and what are the temperatures used in each step? 2) The PCR technique used to measure mRNA abundance is called? 3) The process of producing cDNA from mRNA is called? 4) What is the name of the primer that is generally used to synthesize cDNA from mRNA?

1) -denaturation (95C) -annealing (50-60C) -extension (72C) 2) RT-PCR 3) reverse transcription 4) oligo-dT

1) Describe cloning plasmids and give an example. 2) Describe expression plasmids and give an example. 3) Describe reporter plasmids and give an example.

1) -for isolation and amplification of DNA sequences -contains (i) origin of replication, (ii) selectable marker and (iii) MCS -example: pBluescript (also has blue/white selection for plasmids without/with inserts) 2) -for the expression of proteins in bacteria, yeast, plant or animal cells -contains promoter appropriate for target cell located upstream of MCS -also contains elements found in cloning vector (for cloning, amplification of E.coli) -example: pCMV, a mammalian cell expression vector 3) -for the measurement of gene regulatory sequences -contains a reporter gene that when expressed can be quantitatively measured -MCS is located upstream of the reporter gene -also contains elements found in cloning vector -example: pGL3 (contains the luciferase reporter gene)

1) Describe the detection step for Western blotting 2) Describe the various elements involved in detection

1) -membrane with protein bands is incubated with an antibody specific for the protein being studied -antibody will bind only to the protein band it is responsible (eg anti-tubulin antibody binds only to tubulin protein band but not beta globulin, actin, etc) -membrane-bound antibodies can be detected using radioactive, calorimetric or fluorescent labeling techniques 2) -Enzyme=horse radish peroxidase enzyme -Substrate=luminol -tubulin band; multiple tubulin molecules in same band -primary antibody; the anti-tubulin antibody bound to tubulin band -secondary antibody; bound to primary antibody -enzyme: converts substrate to chemi-luminescence signal (light); bound to second antibody -light produces silver grains in Xray film

1) In PCR methodology, what are the components of the reaction mixture? 2) What are the 3 PCR steps and what happens in each one? 3) How often do you repeat these cycles?

1) -target dsDNA -pair of oligonucleotide primers -thermostable DNA polymerase -dNTPs 2) -Denaturation of dsDNA (95C-approx 15 sec; DNA containing sequences to be amplified is denatured at high temperature (separates the DNA strands)) -annealing of primers to ssDNA (50-60C-approx 30 sec; anneal synthetic DNA primers to the denatured DNA strands; temperature is primer specific (dependent on G:C content); approx 5C below the melting temperature of the primers -primer DNA extension (72C-approx 30-90 sec); DNA polymerase replicates the DNA using the free 3'OH provided by the primer and one of the DNA strands as the template; extension time is dependent upon template length) 3) 20-30 times

1) What does a dideoxy nucleotide sequencing reaction consist of? 2) Describe what happens to the fragments that result from the reaction

1) -template DNA -sequencing primer -DNA polymerase -4 different dNTPs mixed with ~1% of 4ddNTPs labelled with different fluorescent dyes (eg red for ddTTP, green for ddATP, blue for ddCTP, dark purple for ddGTP) 2) -the synthesized DNA fragments will be of different sizes, each ending with a specific fluorescently labelled ddNTP -fragments size-separated by gel electrophoresis and exit the gel in increments of single nucleotides (smallest exits first) -fluorescent colour for each size monitored as DNA exits the gel -sequence 'read' by decoding the colour for each fragment, shortest fragment to longest fragment

1) What is structural genomics? 2) What is comparative genomics? 3) What is functional genomics? 4) What is synthetic genomics?

1) -the study of gene structure -genome mapping and sequencing 2) -the study of genome evolution -the comparison of genomes of different organisms 3) -the study of gene function -analysis of the transcriptome and the proteome -uses the information from genome sequencing to describe gene and protein functions and interactions 4) uses information from genome sequencing to create new or modified organisms

1) What are the two steps of the Southern blotting procedure? 2) What is molecular hybridization? What does it require? When does it occur?

1) -transfer and immobilize the DNA fragments from an electrophoretic gel onto a membrane -detect the presence of specific DNA fragments through molecular hybridization 2) the annealing of a DNA strand to its complementary DNA strand -prior denaturation (separation of the two strands of the DNA either by heat or chemical treatment) -renaturation occurs when the temperature is reduced

1) What are the questions asked when using immunofluorescence imaging? 2) Give the steps of immunofluorescence imaging.

1) -where in the cell is the protein localized? -is the location of this protein altered by treatment? 2) -grow cells on a glass slide -fix cells (paraformaldehyde) -block with protein solution -incubate with first degree antibody (detects protein of interest) -wash (remove unbound antibody) -incubate with second degree antibody (detects the first degree antibody) -wash -analyze with a fluorescence microscope

1) What is a comparative microarray? 2) Give the steps of a comparative microarray

1) 2 different samples (each labelled with a different fluorescent dye) are hybridized to one microarray 2) -isolate RNAs from control and experimental cells or tissues -synthesize cDNAs labelled with fluorescent dyes by RT-PCR -mix equal amounts of cDNAs and hybridize to microarrays -record the microarray result with a laser scanner at dye-specific wavelengths and analyze the data with appropriate computer software

1) How are restriction endonucleases named? Give an example. 2) What do restriction enzymes generally recognize? What do they create as a result? 3) What is a palindrome? Give an example.

1) According to the bacterial strain in which it was originally identified; EcoR1: E.coli strain RY13 restriction enzyme number 1. 2) palindromic sequences; either staggered (aka cohesive, sticky) or blunt ends 3) a sequence that reads the same in either direction; the DNA is identical but inverted in a complementary strand

Give the steps of positional cloning of Disease associated genes

1) Construct pedigrees from affected families and collect blood samples to isolate DNA 2) Perform PCR and gel electrophoresis for hundreds of different STRs (or VNTRs, SNPs, RFLPs) that are spread throughout the gene 3) Perform a statistical analysis to determine which STR allele is most likely to be linked with the disease based on the pedigree data (linkage analysis) 4) Narrow in on the genes present in the genome near to that STR (ie by low % recombination between gene and different STRs) 5) Identify gene by positional cloning: characterize contigs located within this region or by chromosomal walking -ask which map markers (STRs, RFLPs) are closely associated with the disease (mutant) phenotype -candidate genes are sequenced in wild-type and disease (mutant) individuals to search for mutations -complementation tests can be performed to see if the wild-type gene restores function in cell lines from individuals with the mutation

1) Detection of VNTR or STR polymorphisms by PCR and southern blotting/capillary gel electrophoresis is called? 2) The resulting specific banding patterns are called? 3) What do the individual bands of DNA fingerprints represent? 4) How does the probability of identical fingerprints between two individuals (except identical twins) change as more polymorphisms are used? Example? 5) What are DNA fingerprints used in?

1) DNA fingerprinting 2) DNA fingerprints 3) different alleles of VNTR/STR 4) the probability decreases; eg, for CODIS (combined DNA index system) with 13 STRs the probability is less than 1 in 6 trillion 5) paternity suits and forensics

1) In Northern blotting for RNA analysis, which is the probe and which is the target? 2) What is Northern blotting? 3) How does Northern blotting compare to Southern blotting? 4) Why must care be taken to prevent RNA degradation? 5) What are some applications of Northern blotting?

1) DNA is the probe, RNA is the target 2) the transfer of size-fractionated RNA from an electrophoresis gel to a membrane support 3) similar to Southern blotting, except that the nucleic acids blotted onto the membrane are intact mRNA (not re-digested DNA) 4) RNA is less stable than DNA 5) -detection of the length and type of transcripts from a specific gene -quantitative measurement of the transcription level of a gene

Give the steps of the procedure for a DNA microarray assay

1) DNA microarrays are designed with genes of interest (ie probes) represented on the microarrays as separate spots of ssDNA oligonucleotides 2) DNA (or cDNA for RNA) samples are labelled fluorescently (or radioactively for low-density, membrane-based arrays), and hybridized to the microarray surface 3) After extensive washing of unbound labelled DNA, spots with bound DNAs are detected by fluorescence (or autoradiography) 4) positive spots correlated to spots of specific oligonucleotide probes (each spot represents a different gene) 5) for comparitive transcriptomics (eg treatment with/without hormone) use two different fluorescently labelled samples (different colours) and compare their relative intensities

1) What happens during genetic selection? 2) Describe an example of cloning by complementation

1) DNA sequence restores the wildtype phenotype to a mutant organism 2) -isolate mutant bacterial strain that has lost its ability to grow without glucose -transform cells with a genomic library or cDNA library from wildtype cells -screen for colonies that grow in the absence of glucose -these cells have taken up a plasmid that encodes a wild-type version of the mutant genomic gene (you have isolated a gene required for glucose metabolism)

1) What are satellite DNAs? 2) What are minisatellites and now long do they tend to be? 3) What are microsatellites and how long do they tend to be? 4) What is this form of DNA polymorphism due to? 5) What is a highly polymorphic gene marker? 6) What are VNTRs and STRs readily analyzed by?

1) DNAs with repetitive sequences 2) Variable Number of Tandem Repeats (VNTRs); ~10-80 nucleotides long 3) short tandem repeats (STRs); ~1-10nts long 4) the variable number of the nucleotide sequence repeats (a few repeats to hundreds of repeats) between restriction enzyme sites 5) when a population has many different numbers of repeats in a given locus 6) PCR or southern blotting; they are hypervariable regions

What are the applications of Southern blotting? Describe each.

1) Detect the presence of a DNA fragment in genomic DNA -probe a DNA library with radioactively labelled DNA to identify clone containing a sequence homologous to the probe 2) Detection of homologous DNA (similar DNA sequences in different species that have been conserved during evolution) 3) RFLP (Restriction Fragment Length Polymorphism) -paternity testing, genetic mapping -detects number of RE fragments and their sizes for sequences that are highly repeated in the genome 4) Detection of gene variants indicative of disease -Huntington's disease patients have one normal and one larger Huntington gene

What are the major steps in the construction and screening of DNA libraries and what happens in each?

1) Digestion of vector -cut vector with restriction enzyme within the multiple cloning site 2) Digestion of genomic DNA -cut with same restriction enzyme used to cut vector 3) Annealing and ligation -allow the compatible sticky ends of vector and target DNAs to base pair with each other -treated annealed DNA with ligase to seal the 'nicks' (broken phosphodiester linkage between two adjacent nucleotides on the same DNA strand) 4) Transformation -of E.coli (transfer the DNA into E.coli cells) 5) Selection -grow the transformed cells on selective media appropriate for the vector (eg selection media containing ampicillin)

1) Who developed the concept of Southern blotting/Southern hybridization)? 2) What three questions can this method answer? 3) Give the steps of the process of Southern blotting/Southern hybridization.

1) Edwin Southern 2) -which region of the genome contains a coding sequence for a specific gene? -how many copies of this sequence are present in the genome? -are there homologous sequences in the genomes of other organisms? 3) -digest genomic DNA with restriction enzyme -separate fragments by gel electrophoresis -transfer fragments onto a membrane -hybridize a radioactive probe DNA to membrane -expose membrane to Xray film

1) What does ENCODE stand for? 2) What are the ENCODE project goals? 3) What is ENCODE?

1) Encyclopedia Of DNA Genetic Elements 2) -to identify all nongenic functional elements in the human genome: promoters, enhancers, silencers, sites of methylation and acetylation -to build a comprehensive parts list of functional elements in the human genome, including elements that act at the protein and RNA levels, and regulatory elements that control cells and circumstances in which a gene is active 3) -an international collaboration of research groups funded by the National Human Genome Research Institute

What are some applications of PCR?

1) Gene cloning -must have the gene sequence in order to design gene specific primers -genebank contains all sequenced genes 2) Gene diagnostics -prenatal diagnosis of inherited human diseases -forensic identification using small amounts of biological samples 3) DNA quantification -measure abundance of a microbe in a sample 4) Measure gene expression -RT-PCR (reverse-transcriptase-mediated PCR) -copies mRNA into cDNA, which can be amplified and quantified 5) Site-directed mutagenesis -allows you to alter the sequence of a gene

1) What are the two options for mapping/sequencing for the Human Genome Project? 2) What are the steps for mapping/sequencing?

1) HUGO (hierarchical, three-stage approach used by the public consortium) and shotgun (Celera) (whole-genome shotgun approach used by Celera genomics) 2) genetic (linkage) mapping (distances in centimorgans) -physical mapping (distances in nucleotide bases) -DNA sequencing

Describe the steps of the construction/screening of a DNA library (mouse example)

1) Isolate E.coli cosmid pJ88 DNA and mouse genomic DNA 2) Cleave cosmid and mouse DNAs with restriction endonuclease EcoRI 3) Mix cosmid and mouse DNAs under annealing conditions and treat with DNA ligase 4) Recombinant DNA containing mouse EcoRI restriction fragment inserted into self-replicating E.coli cosmid DNA

1) Do physical map distances always correlate with genetic distance? 2) How can physical and genetic maps be correlated with cytological maps? 3) What are anchor markers? Example? 4) What are genetic maps okay for? 5) Are physical or genetic maps more accurate?

1) No- because recombination frequencies (cM) are not always proportional to the molecular distances (bp) 2) by in situ hybridization or by association with chromosome banding patterns 3) DNA sequences that are mapped both genetically and physically (for instance, RFLPs) 4) mapping the gene order and relative distance 5) Physical- they are required to determine the location of a gene

1) The process of amplifying DNA from a small amount using primer pairs is called? 2) The name of the enzyme used in PCR for strand elongation (extension) and resistant to high temperatures is? 3) The name of sequence specific single-stranded DNAs used to initiate strand elongation in PCR are called?

1) PCR 2) taq polymerase 3) primers

1) What does PAGE stand for? 2)Describe the steps involved in protein separation and analysis by SDS-PAGE

1) Polyacrylamide gel electrophoresis 2) -cells are lysed in buffer containing the detergent Sodium Dodecyl Sulfate (SDS) -SDS binds proteins imparting a net negative charge, migrates to anode -polypeptides migrate through a porous polyacrylamide gel matrix in an electric field -mobility is a linear function of the logarithms of their molecular mass (smallest polypeptide runs fastest)

Give the very general steps of chromosome walking

1) Prepare a restriction map of the clone 2) subclone B-H fragment 3) screen genomic library with subclone as probe 4) Prepare a restriction map of the new clone 5) subclone H-E fragment 6) Rescreen library with new subclone as probe 7) Repeat steps 1-3 for as many cycles as needed to reach the gene of interest

Give the general procedure of RT-PCR

1) Reverse transcriptase 2) PCR cycle 1 with reverse primer 3) Amplification of cDNA by PCR (many cycles)

1) The technique used to separate proteins by size in a gel matrix is called? 2) The technique used to identify a specific protein band following electrophoresis in a polyacrylamide gel? 3) The technique used to study protein localization in fixed cells is called? 4) What is the advantage of using GFP-tagging as opposed to immunofluorescence imaging to study protein localization?

1) SDS-PAGE 2) Western blotting 3) immunofluorescence 4) GFP-tagged proteins can be visualized in living cells in real time over extended periods of time; immunofluorescence imaging only gives you a temporal snapshot

Give the general steps for the procedure of dideoxy nucleotide sequencing

1) Set up a DNA polymerization reaction containing the following: template strand, primer strand, DNA polymerase, dGTP, dATP, dCTP, dTTP, and -all four 2',3'-dideoxyribonucleoside triphosphate chain terminators, each labelled with a different fluorescent dye: ddGTP, ddATP (green), ddCTP (blue), and ddTTP (red) -the dNTPs are in excess relative to the ddNTPs 2) Incubate reaction mixture. Synthesized chains terminated with: ddG, ddA, ddC, ddT 3) Denature products and separate by polyacrylamide capillary gel electrophoresis 4) Record sequences with a scanning laser, a fluorescence detector, and a computer -the obtained DNA sequence is the complementary strand of the original DNA template strand

1) What does SNP stand for and what is it? 2) Where are most SNPs found? 3) Between two individuals, there is an SNP in every _______ base pairs, on average? 4) How can SNPs be identified and detected? 5) What is a haplotype?

1) Single nucleotide polymorphism: genetic polymorphic marker; it is a single base pair substitution with a reference sequence (ie A/T to G/C or G/C to A/T) 2) noncoding regions 3) 1200 4) by DNA sequencing, and detected using gene chip technology 5) a set of SNPs on the same chromosome that tend to be inherited together

1) Who discovered restriction enzymes? 2) What do restriction enzymes do for bacteria and how? 3) How is the bacterial genome protected from restriction enzyme degradation? What is this catalyzed by?

1) Smith and Nathans 2) protect genetic material of bacteria from invasion by foreign DNA (viruses) by restricting their intrusion 3) by methylation of nucleotides within the sequence recognized by the restriction enzymes; specific methylases after DNA replication

1) What is a cytological map? 2) What are physical maps? 3) What are genetic maps based on? How can you determine recombination frequencies?

1) a banding pattern of stained chromosomes 2) when molecular biology techniques are used to arrange sequence features on a linear map 3) -the use of genetic techniques to construct maps showing the relative positions of genes and other sequence features in the genome -by cross-breeding or pedigree analysis (linkage analysis)

1) What is the Sanger dideoxy chain termination method? 2) What are the steps of this process?

1) a method for determining the nucleotide sequence of a DNA molecule 2)-DNA synthesis requires a template and a primer with a free 3' hydroxyl -polymerization proceeds in the 5' to 3' direction -the 3'OH is attached to the alpha phosphate of the incoming nucleotide (the 3'OH is required for chain elongation) -the 2'3'dideoxyribonucleoside incorporation terminates DNA synthesis (there is no longer a 3'OH)

1) What are contigs? 2) What do they usually begin as? 3) What is the general process? 4) What are some strategies to order genomic clones into contigs?

1) a set of overlapping genomic DNA clones (eg from a 'clone library') 2) YAC clones (yeast artificial chromosome) since they can hold 200-500 kilobases- then subclone smaller fragments into general purpose cloning vectors 3) create a restriction map or DNA sequence of each clone -overlapping clones (contigs) identified by overlapping restriction map patterns and sequences 4) -restriction mapping -DNA sequencing -STS (sequence-tagged site) mapping

1) What is Huntington's Disease? 2) What is the Huntington gene? Mutant Huntington gene? 3) What is HTT important for? 4) What does mHTT cause? 5) What is the difference in the progression of the disease for a person homozygous HD vs heterozygous? 6) What is a gain of function mutation? 7) Why is mHTT a mutant?

1) an autosomal dominant disorder (mHTT or HD) 2) Htt; mhtt 3) transcription, cell signalling, and 4) progressive degeneration of the central nervous system beginning at age 30-35 resulting in death ~10-15 years after appearance of symptoms 5) same age of onset, but more rapid degeneration for a person homozygous 6) where the mutant form of a protein has a different effect than the normal protein (ie Huntington's Disease) 7) it cannot fold properly, forming aggregates that are neurotoxic

1) How can you position genes on chromosomes? By using what? 2) What is the general process of using DNA probes for in situ hybridization? 3) What are the steps of in situ hybridization?

1) by in situ hybridization; by using either radioactive DNA probes (detectable by photographic film) or fluorescent DNA probes (detectable by fluorescence microscopy) 2) -hybridize radioactive (eg 32P) or fluorescent DNA probe to metaphase cells on a glass slide -autoradiography (or fluorescence microscopy) -microscopic observation 3) -squash cells on slide -treat with 0.07N NaOH for 2 min -incubate with radioactive DNA, then wash to remove unhybridized single strands of DNA -coat slide with emulsion, expose, and develop autoradiograph

1) What does each bacterial colony contain? 2) How many recombinant clones are typically in a library? 3) What happens to each bacterial cell during growth?

1) clones of cells each replicating a plasmid containing a unique fragment of DNA from the genome used to construct the library 2) thousands 3) each bacterial cell will produce multiple plasmids per cell allowing for high level plasmid amplification

1) What are cDNA libraries? 2) Give two elements of cDNA libraries 3) How do you make cDNA libraries? (4) 4) What does cDNA stand for?

1) copies of all mRNAs in a cell 2) -cannot clone mRNA directly (has to be in DNA form) -cDNAs contain DNA sequences complementary to the mRNAs expressed in the cells used to contruct the library 3) -isolate mRNA from cells -convert mRNA into cDNA (requires the enzyme reverse transcriptase (RT)) -ligate cDNA into cloning vector -transform bacteria 4) complementary DNA (ie complementary to mRNA)

1) What are restriction endonucleases? 2) What do they do (or cut) specificically? 3) What happens to the ends of DNA cut by restriction enzymes?

1) enzymes that cut at specific sequences (restriction sites) within DNA molecules 2) they break phosphodiester bonds that link nucleotides together 3) have single-stranded overhangs; ends are cohesive (complementary, sticky)- they can reanneal

1) What does the mhtt mutation result from? 2) How many CAG repeats do normal individuals have? HD patients? 3) What is the repeat number in mhttt correlated with? 4) Which amino acid does CAG encode? 5) What does HTT generate? What happens if there is excess of this?

1) expansion of a trinucleotide repeat, CAG with the htt (huntington) gene 2) 11-34; 42 to >100 copies 3) age of onset and severity of degeneration 4) Q (glutamine) (Gln) 5) a polyglutamine tract in huntington protein; the folding properties of mHTT are altered; misfolding leads to mHTT aggregation, loss of neurotransmission and atrophy of neuronal tissues

1) What does FISH stand for? 2) What are the steps to this process? 3) Describe an example with telomeres

1) fluorescent in situ hybridization 2) hybridize biotin-bound DNA probe to cells on a glass slide -incubate with avidin bound to a fluorescent dye -microscopic observation with fluorescence microscope 3) -telomeres detected by FISH -telomeric DNA-biotin probe bound to avidin conjugated to a yellow fluorescent dye -DNA stained red with a DNA specific dye

1) How does the shot-gun method/"let the computer do all the work" method work? 2) What is a problem with this method?

1) genome is randomly fragmented and every fragment is sequenced (usually by "parallel" sequencing methods); no need to clone DNA first -process is repeated to create numerous overlapping sequence reads -sequences are pieced together using a computer that matches overlaps 2) it is difficult to order fragments with repetitive DNA

1) What does it mean to use positional cloning to find a gene? 2) What does positional cloning usually start with? 3) What are the steps that follow this? 4) For functional verification of candidate genes, what do you confirm? 5) What can restore the wild type (healthy) phenotype?

1) identification and cloning of a gene on the basis of its relative position on the chromosome 2) linkage analysis to map the gene of interest to closest genetic markers (eg RFLPs, VNTRs, STRs, SNPs) 3) use association frequency between markers and genes -then do a comparison of affected and non-affected individuals in pedigrees -next, identify candidate genes with the chromosome region on the physical map 4) that mutation of the candidate gene is responsible for the observed phenotype by comparing healthy vs diseased persons 5) correction of the mutation or introduction of the wild-type gene in a model system

1) What are cloning vectors used for? 2) Give five elements of cloning vectors 3) What are the three essential components of plasmid vectors?

1) isolation and amplification of DNA sequences 2) -mostly originated from bacteria (some from yeast) -double-stranded circular DNA -extra-chromosomal -replicate independent of cell DNA (plasmids can be multi-copy: ie >1 plasmid/cell) -small, up to about 10kb (for easy manipulation; maximum insert size usually approx 15kilobase pairs) 3) -an origin of DNA replication (for amplification in bacterial cells) -a selectable marker: antibiotic resistance gene -at least one unique RE cleavage site outside of the origin and resistance gene (many have multiple cloning sites, MCS)

Briefly summarize each of the following: Cytological Map Genetic Map Physical Map Contig Map Genomic Sequence

1) microscopy of chromosome, staining bands, eg Giemsa(G) or quinacrine (Q) differential staining 2) relative position of genes, RFLP, STR, VNTR based on linkage studies (recombination frequency); about 1Mb (centimorgans) apart 3) relative location of DNA sequences based on RE sites, STS or contig (position) based upon their actual physical distance from each other (~100kb apart) 4) set of ordered overlapping genomic clones (contigs) 0.5 to 1Mb each (eg ordered by RE sites) 5) ordered contigs for each chromosome (measured in base pairs)

1) What are DNA polymorphisms? Used as? 2) What are RFLPs? 3) What is RFLP mapping used to detect and how? 4) What is RFLP also due to? Example? 5) How are RFLPs mapped? 6) As a molecular 'phenotype' what are RFLPs used to classify?

1) nucleotide sequence variations among individuals within a population; used as genetic markers 2) DNA polymorphisms due to addition or deletion of restriction enzyme sites 3) the presence or absence of restriction sites at a genetic locus by restriction digestion followed by Southern blot hybridization 4) variations in lengths of restriction fragments, eg variable numbers of repeat elements, indels (insertions and deletions) within a fragment 5) like other genetic markers 6) the progeny of crosses as parental or recombinant

1) What is used as a hybridization probe for DNA bound to a nylon membrane? 2) Give the steps for colony hybridization

1) radioactively labelled DNA or RNA 2) -replica plate bacterial colonies from a genomic library onto a nylon membrane -lyse cells and denature the double-stranded DNA -add radioactive cDNA (prepared by reverse transcription of mRNA in the presence of radioactive nucleotides) and incubate to allow hybridization of probe to membrane bound DNA -wash to remove unbound probe -expose membrane to Xray film--> probe bound to membrane will react with silver grains in the film--> when developed these will produce dark spots -use location of radioactivity on film to identify positive colony from the original plate

1) What are genetic maps based on? 2) What is the resolution of genetic maps like? What is a centimorgan? 3) What are some common genetic maps? Example? 4) What is a physical map based on? 5) What is the resolution of a physical map like? 6) What are some common physical maps?

1) relative position of linked genetic markers that are polymorphic (different phenotypes) 2) it's low: 1cM~1mb (1 megabase=1 million base pairs); 1 centimorgan (cM): distance that yields a recombination frequency of 1% 3) genotypes, RFLP, mini-(VNTR) and micro-(STR) satellites, SNPs; in drosophila red eyes vs white eyes 4) physical non-polymorphic (sequence specific) markers: actual molecular distances measured in base pairs 5) High (in 100kb or smaller) 6) -contig map (ordered DNA fragments mapped by RE sites or DNA sequence) -restriction map (relative location of restriction enzyme sites) -STS (sequence-tagged site) map

1) When ordering contigs by restriction mapping, what do the vertical bars represent? 2) Describe the steps of the process of ordering contigs by restriction mapping

1) restriction enzyme cleavage sites on the DNA for one restriction enzyme 2) -each clone must be cut with several different restriction enzymes -the sizes of each of the fragments produced in single versus combination digests is used to construct a map for each clone -computer-based alignments are used to order the clones

1) What is the concept of measuring gene expression by RT-PCR? 2) Give the steps of RT-PCR 3) What are some applications of RT-PCR?

1) reverse-transcriptase mediated PCR -copies mRNA into cDNA, which can be amplified and quantified 2) -isolate mRNA from different sources or treated samples -reverse transcription: single-stranded mRNA is reverse-transcribed to single-strand complementary DNA (cDNA) using oligo dT or gene specific primers and a viral reverse transcriptase (RNA dependent DNA polymerase) -PCR amplification of the cDNA to dsDNA using forward and reverse gene specific primers -compare the quantity of PCR products (semi-quantitative; real time PCR) 3) -detect and quantify the amount of a specific mRNA in different samples (determine the level of transcription of a particular gene) -clone a gene of interest by creating and amplifying cDNA

1) What does STS stand for? What are they? 2) What does EST stand for? What are they? 3) What do ESTs represent? 4) What can ESTs be used for?

1) sequence tagged site; short, unique genomic sequences, ~200-500bp identified by whole genome sequencing 2) expressed sequence tags: short (usually <1000bp) single pass squence reads from mRNA (cDNA) sequences; typically produced in large batches; tags (some coding, others not) of expression for a given cDNA library 3) portions of cDNAs generated by sequencing cDNA fragments; a snapshot of genes expressed in a given tissue and/or at a given developmental stage 4) gene mapping -studying transcript and single nucleotide polymorphisms -discovery of putative gene products -comparison of gene expression patterns -probes for microarrarys/gene chips

1) In the second cycle of PCR, all four double-stranded DNA molecules are..? What does this mean?

1) template copies -one strand on each contains DNA that is outside of the boundary defined by the primers

1) What does the bluescript plasmid express? 2) Where is the MCS located? 3) What does insertion of foreign DNA into the MCS do? What is this called?

1) the E.coli lacZ gene (encodes beta-galactosidase) 2) within the plasmid encoded lacZ gene (actually a portion of lacZ) 3) it disrupts the reading frame of the lacZ gene; "insertional inactivation"

1) What is the transcriptome? 2) How can transcriptomes change? 3) What is a proteome? 4) What is proteomics? 5) What is interactomics?

1) the entire set of mRNA transcripts produced by a given organism 2) -with development, time, tissue, extracellular signals, environmental insults, virus infection 3) -the entire array of proteins encoded in an organism 4) the study of the proteome 5) study of all protein-protein interactions

1) Knowing the sequence of all human genes makes it possible to study what? 2) Give the steps of the process of using DNA microarrays to study gene expression 3) What will this tell you?

1) the expression of large subsets of genes in a single experiment 2) -DNA probes (complementary to RNA transcripts) can be synthesized corresponding to every known gene -probes can be bound in an array at high density on solid supports -isolated mRNA can be reverse-transcribed into cDNA using fluorescent nucleotides -fluorescently labelled cDNAs can be hybridized to these microarrays and the bound DNA on the array quantified 3) -which genes are expressed in a sample -the relative abundance of transcripts between two samples

1) What are expression plasmids designed for? 2) What must they be able to do? 3) What must they contain (3)?

1) the expression of proteins in bacteria, yeast, plant, or animals cells 2) replicate in bacteria in order to amplify the plasmid 3) -a selectable marker to identify bacteria containing the plasmid -a cloning site located downstream from a promoter appropriate for the host cell to allow the inserted gene to be expressed -a selectable marker (antibiotic resistance gene) appropriate for the host cell to allow for selection of cells that have incorporated the plasmid in their genome

1) What does MCS allow for? 2) What do reporter genes code for? 3) What are reporter genes used for? 4) What does luc+=? 5) What is done to the luciferase protein? (2) 6) What is the luciferase protein proportional to? 7) Does the reporter gene have a promoter to control its expression? 8) What happens at the MCS? 9) What is the reporter gene called in this example?

1) the insertion of potential gene regulatory sequences 2) a protein that can be visualized or measured 3) to study gene regulatory sequences after transfection into cells 4) the firefly luciferase gene 5) -it is assayed (converts luciferin to light) -that light can be measured in a luminometer 6) [light] proportional to [luciferase protein] proportional [luciferase mRNA] which is proportional to transcriptional activity 7) No 8) insert gene regulatory sequences here 9) firefly luciferase (luc)

1) What is DNA and RNA electrophoresis? 2) To which electrode will DNA migrate and why? 3) What are their rates of migration inversely proportional to? 4) What are some applications of DNA electrophoresis?

1) the separation of a mixture of DNA (or RNA) fragments in a porous gel (agarose or polyacrylamide) in an electric field 2) toward the anode (positive electrode) , because DNA is negatively charged 3) their size (ie the shorter ones move faster than the longer ones) 4) -determine size of DNA fragments -isolate DNA fragments for cloning -in preparation for Southern/Northern blotting

1) What is genomics? 2) What is metagenomics? 3) What is transcriptomics? 4) What is proteomics? 5) What is metabolomics? 6) What is interactomics?

1) the study of the 'genome', the entire DNA genetic material of an organism 2) study of aggregate genomes from a complex of organisms (eg from a gut microbiome) 3) study of the 'transcriptome', all RNAs (eg tRNAs, rRNAs, snRNAs, microRNAs, mRNAs) expressed in a cell 4) study of the 'proteome', all the proteins of an organism and their interactions 5) study of the 'metabolome', all metabolic pathways and intermediaries in an organism 6) study of the 'interactome', all interactions, (eg protein-protein, protein-nucleic acid, protein-ligand) of an organism

1) What is genetic mapping of chromosomes based on 2) What is map distance? 3) What is the recombination of markers due to? 4) There is a greater chance of recombination between genetic loci that are..? 5) What are some examples of genetic markers?

1) the use of genetic markers (though they are not necessarily just genes) 2) the frequency of genetic recombination between two loci (markers) on the same chromosome 3) 'crossing over' 4) far apart, compared to those that are closer together (eg distance between gene A/a and gene D/d; A and a are alleles of the same gene on homologous chromosomes, "A" is the normal gene, "a" is the corresponding mutant gene) 5) -genes -RFLP (restriction fragment length polymorphism) sites -minisatellites (variable number tandem repeats; VNTRs) -microsatellites (short tandem repeats (STRs)) -single nucleotide polymorphisms: SNPs

1) What is DNA quantification used for? 2) Describe elements of quantitative real-time PCR

1) to measure abundance of microbe in a sample 2) -a fluorescent dye is incorporated into the amplified DNA -the amount of fluorescent DNA increases exponentially with each PCR cycle -fluorescence can be measured at each PCR cycle -samples with more starting material (target DNA) will produce a fluorescent signal that crosses an arbitrary threshold at an earlier PCR cycle than a sample in which the target DNA is less abundant

1) What is SDS-PAGE used for? 2) How can the protein bands in gels be visualized? 3) What is Western blotting and what is another name for it? 4) How does it detect the target protein?

1) to separate all the polypeptides in a complex mixture 2) by Coomassie blue staining 3) transfer of separated proteins from gel to a solid support (membrane); immunoblotting 4) by a specific antibody