Exam 3

Question 1

454 GS20 sequencing. How many reads and how long?

Answer 1

The first next generation DNA sequencing platform. Produces 200,000 sequence reads of 100 bp each.

Question 2

What is Next Generation Sequencing?

Answer 2

Sequences many DNA molecule in parallel. Bypasses the need to individually clone and grow each molecule prior to sequencing.
Good for sequencing complex mixtures of DNA molecules, not for individual plasmids or PCR products. Produces up to billions of sequence reads in one run.

Question 3

compare Sanger vs Next Gen sequencing

Answer 3

Sanger needs to be cloned in vivo and amplified then is cycle sequenced with labeled ddNTPs. Next Gen just ligates adaptors and then generates a polony array that is read with cyclic array sequencing

Question 4

common features of all next generation sequencing

Answer 4

library preparation and clonal amplification (except PacBio or nanopore)

Question 5

describe library preparation for next gen sequencing

Answer 5

RNA or genomic DNA is fragmented and sequencing adaptors are added to the ends of fragments. These adaptors are used for clonal amplification and sequencing

Question 6

describe clonal amplification for next gen sequencing

Answer 6

Two ways:

1. Emulsion PCR: takes place in small droplets in an emulsion with a small bead and one template molecule. result is a bead coated with copies of that one molecule (slide 15)
2. Bridge Amplification: individual molecules are amplified on a substrate coated with oligonucleotides. result is many small spots of DNA, all DNA in one spot is identical. (slide 16)

Very sensitive to amount of input! too much gives mixed sequences, too little gives little data

Question 7

describe 454

Answer 7

Older tech, support ended 2016. Yields up to 1 million reads per run of 1000 bp length. Used mostly for sequencing amplicons (PCR products) from complex samples. Runs take 1 day. cost per base is high compared to other technologies. Has problems with homopolymers stretches

1. Emulsion PCR, beads deposited in plate with small wells
2. polymerase bound to each molecule and NTPs flow across one by one.
3. polymerase adds if it can, light is produced. camera records light production, determines sequence of DNA in each well

Question 8

describe ion torrent

Answer 8

clonal amplification is the same as 454 emulsion PCR. sequencing is same as 454 except detects pH change instead of light production. Yields up to 80 million reads of 200-400 bp length. Cost is much less that 454. Runs very fast (2 per day). Has problems with homopolymers stretches

1. Emulsion PCR, beads deposited in plate with small wells
2. polymerase bound to each molecule and NTPs flow across one by one.
3. polymerase adds if it can, pH is changed. meter records pH, determines sequence of DNA in each well

Question 9

describe Illumina sequencing

Answer 9

Dominant technology right now. Yields up to 20 billion reads of 150-300 bp (depends on instrument). Takes up to 2 weeks. Cost is very low

1. Bridge Amplification
2. Addition of fluorescent nucleotides (one added per molecule)
3. slide is scanned and color of each cluster recorded
4. nucleotides are unblocked
5. repeat addition of nucleotides and detection for up to 300 cycles

Question 10

describe Pacific Biosciences sequencing

Answer 10

Single-molecule real time sequencing (SMRT). No clonal amplification, detects incorporation of fluorescent nucleotides on a single strand. DNA library molecules are circular. reads dozens of kb in length. Used when very long reads are needed, but accuracy is lower. Produces up to 4 million reads of 20,000 bp lengths. Fast, only 4 hours

Question 11

describe Oxford Nanopore

Answer 11

super new technology. DNA strand is pulled through a pore in a membrane and electric current is passed through pore. Changes in current depend on the base passing through the pore. Changes in current are translated into sequence. No cloning, no background noice, length is theoretically unlimited. Number of reads is variable. May be able to read RNA directly (unique!) and can distinguish between modified and regular nucleotides. High error rate!

Question 12

Overview comparison of Next Gen sequencing technologies

Answer 12

454: Emulsion PCR, max 1 million reads, max 1000 bp length
Ion Torrent: Emulsion PCR, max 80 million reads, max 400 bp length
Illumina: Bridge Amplification, max 10 billion reads, max 600 bp length
PacBio: No amplification, max 500,000 reads, max 30,000 bp length
Nanopore: No amplification, max ?? reads, max 200,000+ bp length

Question 13

How are next gen sequence data sets analyzed

Answer 13

Large output file (FastQ common) is not analyzed individually like Sanger, but may be aligned to one another to creat a consensus sequence or to compare to a reference

Question 14

What are barcodes (multiplexing)?

Answer 14

Most next gen sequencing technologies produce more data in one run than is needed for many applications. Barcodes or Indexes allow for multiplexing of samples (running many samples at once). Barcodes are short nucleotide stretches that are added to samples to many samples can be sequenced together and then separated by barcode.

Question 15

draw the structure of a typical completed sequencing template (dual indexed)

Answer 15

P5 tail (for bridge amplification), Index 2, PE adapter, DNA fragment, PE adapter, Index 1, P7 tail (for bridge amplification)

Slide 5

Question 16

how was the human genome project accomplished?

Answer 16

Genomic DNA cloned into many thousand BAC clones. Each clone was sequenced individually via primer walking. Sequenced BACs are assembled using matching sequences at ends.
Took 13 years, $3 billion, and 20 universities in several countries

Question 17

what is shotgun sequencing? How was the human genome sequenced differently with this method

Answer 17

Whole genome is broken into small pieces and sequenced individually. then pieces are put together computationally. This is much faster and took about 5 years (finished around same time as BAC clones were finishing)

Nowadays, when whole genome sequencing is needed, we use shotgun approach

Question 18

De novo vs resequencing

Answer 18

de novo: “from the beginning”. No prior genomic information. Assembly is difficult, data is made into contains (longest possible stretches of assembled data possible) and then aided by mate-pair or long reads.

resequencing: you already have a genomic sequence for the species and data is mapped back to the reference. Used to look for differences from the reference. No assembly required, short reads with enough data to reliably place them in the genome are sufficient

Question 19

What is target capture

Answer 19

sequence a small portion of the genome but the same portion of each sample. Reduces the amount of sequencing required.

1. block fragments with oligos
2. hybridize targets to capture probes
3. incubate with magnetic beads which bind the hybridized fragment

Question 20

describe RNA-Seq

Answer 20

Sequencing of transcribed genes. Can be mRNA or can include small, non transcribed RNA. Gives quantitative information about gene expression in sample

1. Fragment RNA before reverse transcription (most common) or cDNA may be fragmented
2. sequencing adaptors are ligated to ends of fragments
3. library is size selected to get fragments the right size for sequencing
4. library is sequenced
5. Reads are mapped to genome and number of reads for each gene are counted. counts measure expression levels

Question 21

heat maps vs volcano plots

Answer 21

heat maps group samples by similarities. Good for finding patters of similarly regulated genes

Volcano plots show fold change/difference of group means (x axis) vs p-value (y axis). Good for identifying significant genes

Question 22

define metagenomics

Answer 22

Study of genetic material from environmental samples, an entire microbial community can be studied without culturing individual species.
All DNA from sample is isolated and sequenced together, sequences are assembled into genomes to study the species present. May sequence RNA instead for metabolic activity.

Question 23

what is 16S and how is it used

Answer 23

a Ribosomal RNA gene with several hyper variable regions. it can be used for species identification. Instead of sequencing whole genomes, we can focus just on 16S gene.

Primers are designed to match conserved regions on either side of hyper variable region. PCR products from samples are sequenced so species present and relative abundance can be learned.

Question 24

What is genotyping and how is it done

Answer 24

A genotype is the genetic makeup of a sample/individual usually with reference to a specific trait/gene. Samples are genotyped at specific loci (genes) for research or clinical purpose.

Most genotyping is done by PCR and sequencing. Simple genes with a small number of alleles can be Sanger Sequenced, but more polymorphic genes cannot (eg Major Histocompatibility Comples has thousands of alleles). These are amplified and sequenced like 16S so alleles can be determined

Question 25

describe genotyping via GBS or “reduced representation sequencing”

Answer 25

Without PCR. Focuses on restriction fragment sites. Genome is digested with some RE and then adaptors are ligated and library is size selected and sequenced. Selects pieces of the genome where the distance between to RE sites is in the range selected for. Focuses on part of genome around RE sites instead of entire genome, simplified data reduces cost and complexity

Question 26

describe ATAC-Seq

Answer 26

Asssay for Transposase Accessible Chromatin using Sequencing. Transposace inserts sequencing adapters at regions of open chromatin. Good for nucleosome mapping and transcriptional activity

Question 27

describe DNAse I Hypersensitive Site Sequencing

Answer 27

Identifies regions of DNA that have fewer nucleosomes (eg active regulatory regions). DNA is digested with DNAse I (only cutes where there are no nucleosomes) then fragment ends are isolated and sequenced. After sequencing, fragments are mapped to the genome.

Question 28

what is epigenetics

Answer 28

factors that are beyond the genetic code. Modifications that do not change the DNA sequence, but change the function. E.g. methylation, histone modification, regulatory RNA.

Epigenetics may be heritable and are affected by environment/chemical exposure. Abnormal epigenetic may lead to disease

Question 29

what is chromatin? type of chromatin

Answer 29

Chromatin: DNA wound with histones for packing

heterochromatin: tightly wound. less accessible to polymerase
euchromatin: more loose and open. transcriptionally active

Question 30

What affects chromatin packing?

Answer 30

Histone modifications:
Methylation causes transcription silencing. Acetylation adds negative charge and opens chromatin structure, increasing expression

Question 31

what is ChIP

Answer 31

Chromatin Immunoprecipitation. Uses an antibody specific for each histone modification to isolate and study DNA that is associated with each histone. Use qPCR to look at changes at specific site or sequence to look at genome wide changes.

Question 32

describe CpG methylation

Answer 32

Cytosine may be methylated. CpG (CG in DNA sequence) islands are regions that have more CpG dinucleotides than expected and are often found in or near promoter regions. Methylation can occur at these islands and leads to gene silencing

Question 33

what are methyltransferases? Name a few key ones

Answer 33

Methyltransferases methylate unneeded genes as cells are specified and differentiate, which is crucial for cell identity/differentiation.
DNMT: Dimethyl Nucleotide Methyl Transferase
DNMT3a/b: lays down initial methylation pattern for cell identity/differentiation
DNMT1: methylates the other C when it find hemimethylated CpG. maintains the methylation pattern after cell division (heritable!)

Question 34

what are imprinted genes

Answer 34

Genes which are differently methylated depending on which parent they come from. Maternal and paternal alleles will be differently expressed. Only a small number of genes are imprinted. Imprinted genes are often found in clusters

Question 35

what is MeDIP

Answer 35

Methylated DNA Immunoprecipitation. just like ChIP, but with antibody for 5-meC. May sequence the pulled down DNA to look for global differences in methylation patterns or use qPCR to look for differences in how much a specific sequence is methylated. Provides information on relative methylation levels at specific regions, but NOT at single base resolution

Question 36

what is bisulfite sequencing

Answer 36

Sodium bisulfite dominates cytosine to create uracil. 5-meC is not converted. This provides methylated data at a single base resolution. U = unmethylated and unchanged C = methylated

note that bisulfite causes lots of DNA breaks and converted DNA is very AT rich. For analysis of target, PCR to amplify and then sequence. Primers must be very long and match converted sequence, avoiding CpG dinucleotides which may be converted

Question 37

describe nanopore detection of m5C

Answer 37

Methylated C residues yield a slightly different signal on Oxford nanopore sequencer so m5C and C can be differentiated with no bisulfite conversion required

Question 38

describe PacBio detection of m5C

Answer 38

Bases are called when labeled nucleotides remain in the volume of detection for an extended time during incorporation. Methylation on the template nucleotide changes the kinetics of the polymerase where there is a longer length of time to call a m5C base than a C base

Question 39

bioinformatics vs computational biology

Answer 39

bioinformatics: biological problems posed by the assessment or study of biodata are interpreted and analyzed. Develop things to store or analyze data related to biology

computational biology: concerned with solutions to issues raised by bioinformatics. Builds models to assist with modeling and understanding biological systems

Question 40

common features of sequence analysis pipelines

Answer 40

Data QC: check for quality of sequence data and remove poor quality reads/bases (Phred scores)
Normalization: accounts for differences in library size
Alignment/mapping: de novo assembly or mapping to a reference. read clustering/dereplication sometimes

Question 41

what are Phred scores

Answer 41

Measure of the quality of the base call. Essentially a score of the reliability of the base call. expressed as ASCII characters. FastQC is a good first look at sequence quality that I think uses Phred scores?

Question 42

describe normalization step in bioinformatics

Answer 42

Necessary in read counting applications in order to compare samples. Samples will differ (sequencing depth, levels of gene expression). There are many methods to do this step (none that I need to know?)

Question 43

overview of common file formats

Answer 43

FASTA: basic sequence information in text format
FASTQ: sequence information with quality scores
SAM/BAM: sequence information with quality scores, can be aligned to a reference
VCF: sequence variant information, compare to other reference
GFF: sequence features such as introns, exons, etc
BED: sequence features and intervals, entire genome

Question 44

why do in vitro mutagenesis?

Answer 44

Used to purposefully change genetic information. Analysis of the resulting changes in gene expression and products helps elucidate the functional effect of the mutation. Can do either specific changes in a known sequence (site directed mutagenesis) or create a wide variety of individual mutations (Random mutagenesis)

Question 45

factors that can effect the success of in vitro mutagenesis

Answer 45

Technique used for constructing mutated DNA
Primer quality
Appropriate expression vector
Effective purification
Development of an assay for detection

Question 46

general features of site directed mutagenesis

Answer 46

Creates a mutation at a defined site and requires a known template sequence. Useful to study the impact of a sequence change (SNPs), to insert or delete a sequence element, or to screen a variety of mutants to determine the optimal sequence.

Typically performed using PCR by introducing the mutation in one of the primers. Resulting PCR product incorporates the mutation very efficiently.

Question 47

describe Primer Extension process and uses

Answer 47

Incorporation of mutagenic primers in independent, nested PCRs to combine them in the final product (essentially can insert mutation in the middle of a gene).
uses flanking primers on either end of the target sequence plus two internal primers that contain the mismatched or inserted bases and hybridize to the region where to mutation will occur. see slide 9 for image

Question 48

describe Inverse PCR

Answer 48

uses primers oriented in the reverse direction to amplify a region of unknown sequence. Can be done in a plasmid without the need to re-clone afterwards! Must use high fidelity polymerase and leave blunt ends for ligation. Can do deletions, substitutions, and insertions and still leave the plasmid construct complete. see slide 13

Question 49

descrive cassette mutagenesis

Answer 49

replaces a section of DNA sequence with a DNA containing the mutated sequence. Insert and target fragments generated by restriction digestion, PCR amplification, or commercial synthesis of products.
Very efficient and provides easy way to screen mutants (sequencing) and flexibility to perform many different mutagenesis events on same vector (multiple cassettes inserted throughout vector).
But, need to identify or insert restriction sites and mutations must be made with primers, cloned, and sequenced before insertion back in plasmid. can lead to occasional double mutants or deletion mutants from primer impurities.

Question 50

Summary of in vitro site directed mutagenesis options and preferred uses

Answer 50

PCR: limited base identity changes at the end of the desired sequence. 5’ or 3’ terminal insertions <100 bp
Primer Extension: internal limited non-random base identity changes, insertions >100 bp, deletions <50 bp, deletions >50 bp if genomic or cDNA template
Inverse PCR: insertions >100 bp if cloned template, deletions <50 bp if cloned template, deletions >50 bp if cloned template

Question 51

Overview of Random mutagenesis

Answer 51

creates mutations at undefined sites and does not require knowledge of sequence or function. Useful to identify protein variants with desired properties e.g. reveal function of proteins by mapping enzymatic active sites, study their role in cellular events, examine structure-function relationships, or understand interaction of genes in biological pathways.

Most commonly done with error prone PCR, PCR with degenerate primers, UV irradiation, and much more

Question 52

describe Error prone PCR

Answer 52

standard method used to create libraries of mutations within single genes. Easy to perform and most commonly used for examining a small number of mutations to identify a desired phenotype. relies on DNA polymerases not being 100% efficient, error rate is increased by high Mg2+ concentration, unequal dNTP concentrations, using a mutated polymerase, Mn2+ instead of Mg2+

Question 53

advantages and disadvantages of Error Prone PCR

Answer 53

advantages: ability to repeat mutagenesis through many rounds of selection. create mutant libraries from randomized cloned genes to screen for specific phenotype
disadvantages: Taq polymerase is biased toward inducing mutations in A and T bases. excessive altered conditions can result in poor amplification and undesired amplicons

Question 54

describe PCR with Degenerate Primers

Answer 54

collection of mutagenic primers are synthesized by including low concentrations of the three non wild type nucleotides at each step of the process. Results in predictable rates of misincorporation per nucleotide and can contain both regions of wild type and degenerate sequences.

Question 55

advantages and disadvantages of PCR with Degenerate Primers

Answer 55

Advantages: most cost-effective method for performing saturation mutagenesis because of the shorter primers used. good for identifying single base changes that alter function. more precise control over location and rate of mutagenesis

Disadvantages: can bias mutations toward sequences with a higher binding affinity for the degenerate primers. changes are limited to the primer binding locations. it is often hard or impossible to select for changes that will result in a subset of amino acids

Question 56

describe chemical mutagenesis

Answer 56

Ability to induce changes in living cells across the entire genome (in vivo!). Avoids the DNA polymerase bias toward AT mutations and allows ability to select for non-lethal mutations since cells must replicate for mutation to be observed.

Ethyl methane sulfonate (EMS): results in GT mismatches. Degree of mutagenesis altered by changing EMS concentration, incubation time and temperature, pH

Question 57

define transfection and its relevance to altering genomic DNA

Answer 57

Transfection is the process of inserting DNA into a eukaryotic cell. Transfected DNA can be transcribed and can produce a gene product. In a few cells, the transfected DNA will be fully integrated into the host genome (randomly! not site specific)

Question 58

what is random integration

Answer 58

Plasmids used during transfection have selection markers for stably transfected cells. Success can be improved by linearizing DNA.
Very easy and fast, fine if you just want to express something
Not site-specific, so can’t be used to change genes. concatamers of the insert are common. may disrupt endogenous genes. expression of insert may be significantly affected by insertion site

Question 59

what is homologous recombination

Answer 59

see slide 5 for visual. Inclusion of targeting sequence can direct DNA insert to a specific site in the genome, sequence is typically several hundred bp on either side of target site. This allows targeting of genes to disrupt or otherwise alter endogenous genes. It is unlikely to be successful and you need to screen for desired cells

Question 60

how do you select for homologous recombinants

Answer 60

Using two selection markers:
Neo is included in the inserted sequence, so all recombinants (random and homologous) can be selected. Neo = antibiotic resistance
HSK-tk is outside of the targeting sequence so it is excluded in homologous recombination events. HSK-tk = susceptibility to antibiotic
slide 6 for visual

Question 61

what is a knockout mouse? how is it made? limitations?

Answer 61

A knockout mouse is a mouse homozygous for a nonfunctional version of a gene. This is helpful for seeing what that gene does. Made by creating one mutated chromosome in ES cells and inserting into a blastocyst stage embryo so the offspring is heterozygous. Breed until homozygous. limitation: only works on mice

Question 62

what is somatic cell nuclear transfer? how is it done? limitations?

Answer 62

Cells from animal to be cloned are taken from tissue and maintained in lab. Nucleus is removed and fused with a donor egg cell lacking a nucleus. The reconstructed embryo grows for 7 days and is implanted into surrogate mother. Cloned animal is born with exact same DNA as tissue donor.
Limitations: usually does not work, only 2-5% success rate

Question 63

describe the general principle behind genome alteration/editing

Answer 63

DNA is cleaved at specific site and subsequent DNA repair may change gene sequence. It can delete a few bases and inactivate the gene, or if a template is added, it can be used by the repair mechanism to alter the targeted gene

Question 64

explain Non-Homologous End Joining (NHEJ)

Answer 64

Mechanism of repair of double strand breaks without template. DNA breaks, ends are processed to remove 5’ and long 3’ overhangs, gap is filled in, ligation to seal

Results in variable deletions and insertions

Question 65

explain Homology Directed Repair

Answer 65

Homologous stretches of DNA can be used to repair double strand breaks to match the template. It may come from the other chromosome or from DNA introduced experimentally. DNA is broken, many factors assemble, the homologous strand pairs and exchanges DNA strands, DNA is extended and sealed together. Many possible combinations of original strand and homologous strand exist

Allows for precise insertion or modification if using an introduced template

Question 66

What are zinc fingers? how are they used in genome editing?

Answer 66

Zinc fingers are common binding sites for transcription factors. Typically there are three fingers, each contacting 3 nucleotides so the sequence of DNA binding side is determined by specific amino acids in the finger. Individual domains can be customized to the DNA binding site and then FokI cleavage domain linked so that you have a custom restriction endonuclease. There is a preference for G rich binding sites (can’t target any sequence)

Question 67

what are Transcription Activator like Effectors and TALENs?

Answer 67

Transcription Activator like Effectors are proteins that bind DNA and activate expression of genes. Contains a central repeat domain with repeats of a DNA binding domain, it’s 34 residues long and residues 12 and 13 are hypervariable and specify identify of DNA recognized.

TALEN is a TAL Effector Nuclease. An engineered protein with the 34 residue repeat made to specify a recognition sequence and fused to a FokI nuclease domain

Question 68

describe CRISPR

Answer 68

From bacterial immune system, CRISPR loci are transcribed and cleaved into short pieces of crRNA which complexes with tracrRNA and CAS9 nuclease to guide Cas9 to target DNA for cleavage. Naturally, the tracrRNA and crRNA are combined into a guide RNA, but a synthetic guide RNA sequence can be made to direct Cas9 to any desired sequence

Question 69

compare CRISPR, TALEN, and ZFN

Answer 69

CRISPR: uses RNA to recognize DNA. Not methylation sensitive. High potential for off target effects. Very easy to design and only requires simple adaptor cloning of 20 bp oligo to target gene

TALEN: uses protein to recognize DNA. Methylation sensitive. Off target effects not well reported. Technically challenging design due to extensive identical repeat sequencing

ZFN: uses protein to recognize DNA. Methylation sensitive. Has off target effects. Design requires customized protein component for each gene sequence

Question 70

What is southern blotting? General procedure?

Answer 70

Method for detecting specific DNA sequences in the genomic DNA of an organism.

1. DNA is cleaved with RE
2. Electrophoresis to separate by fragment size
3. Immobilize by transfer to nitrocellulose or nylon filter by capillary transfer or electroblotting
4. hybridize filter with labelled DNA probes and visualize to see hybrid DNA

Question 71

describe capillary transfer immobilization

Answer 71

slide 5. Gel with DNA fragments is placed on a plastic tray with buffer fed up through a wick. On top of the gel, the membrane (nitrocellulose or nylon) is placed. Then filter paper and tissues and finally a glass plate with weight on top completes the set up. The buffer is drawn up through each layer via capillary action and as it moves up, the DNA is moved up from the Gel to the Membrane
Then fix DNA to membrane by Baking 80 C or UV cross linking

Question 72

describe Electroblotting immobilization

Answer 72

much faster than capillary action. The set up has a negative plate on bottom, a sponge, filter paper, the gel with DNA fragments, the membrane, more filter paper, sponge, and a positive plate. Current flows from the negative plate to the positive plate and draws the DNA from the gel to the membrane. Be careful not to ru to long because DNA can migrate out of the membrane and be lost
Then fix DNA to membrane by Baking 80 C or UV cross linking

Question 73

What are probes?

Answer 73

Labeled nucleic acids complementary to the sequence that is to be detected. Used in hybridization based techniques to locate and bind nucleic acids with complementary sequences

Question 74

How is a label introduced on a probe? 5 ways

Answer 74

End-labeling: T4 polynucleotide kinase transfers Pi from ATP to nucleotides
Random priming: random hexameters prime DNA synthesis randomly on single stranded DNA. requires primer and template, DNA polymerase I or Klenow fragment
Nick translation: treated with DNase to make nicks. Polymerase removes more bases by nick and replaces with labeled nucleotides
In vitro transcription: makes RNA probe by RNA polymerase from bacteriophage. Promoter sequence for polymerase needed upstream of probe sequence. includes labeled nucleotides
PCR with labeled primers or labeled nucleotides

Question 75

types of probes and label location considerations

Answer 75

Types of probes:
single stranded: ready for immediate use
double stranded: must be denatured
Radioactive: usually 32P, but other isotopes can be used. detected via x ray oil,
Nonradioactive: Hapten labels such as biotin or digoxigenin. require secondary detection such as HRP or AP

End labeled is one label per probe. Internal label is at multiple sites and has higher activity

Question 76

Explain random integration and Klenow fragments

Answer 76

Klenow fragments lack the 5 to 3 exonuclease activity of DNA polymerase one and so avoid the loss of incorporated labels. High are of incorporation of radiolabed nucleotide is possible and high DNA probe specific activities.

Question 77

How to perform hybridization and washing (blotting steps)

Answer 77

Add blocking agent (milk, SDS) to prevent non-specific interactions between probes and membrane. Volume exclusion agents increase rate and level of hybridization.
Wash blot with increasing stringency:
Low stringency = high salt, low temp = probe binds to mismatched sequences
High stringency = low salt, high temp = probe binds only to fully complementary sequences

Question 78

How to detect radio labeled blots

Answer 78

P32 is a high energy beta particle emitter and provides good sensitivity for detection of hybridization between the probe DNA and target DNA. Detect with Autoradiography (X ray film) or Phosphorimager.
Phosphorimager has phosphor coated plates store the energy of the radioactive particle which is excited by lasers and releases photons of light. Light is collected and represented as picture. Has greater dynamic range and faster than film

Question 79

How to detect non-radiolabeled blots

Answer 79

Digoxygenin or Biotin is attached to the probe. Then another molecule (HRP or Streptavidin-AP) which is conjugated with an enzyme binds. The enzyme is detectable.
Biotin labeling is made by Klenow Fragment and random hexameters in presence of biotin-dUTP. Streptavidin-AP binds irreversibly to biotin labeled probes. May be detected using chromogenic substrate or chemiluminescent reaction

Question 80

what is northern blot

Answer 80

Same as southern blot, but RNA instead of DNA. Data obtained can tell you the size of transcripts (splice variants, gene length, etc) and expression level of a gene (intensity of band correlates with amount of transcript present)

Question 81

what is western blot

Answer 81

Blot for proteins. Protein is transferred to membranes (same as southern) and detected by probing with antibodies. Antibody binding is detected by another antibody that has enzyme (HRP) or radioactivity

Question 82

what is southwestern blot

Answer 82

Combination of wester and southern, examines binding of proteins to specific DNA sequences. Proteins are run on gel and transferred to membrane then renatured by removal of SDS. Membrane is probed with labeled oligonucleotides or other DNA probes with potential binding site

Question 83

what is dot/slot blot

Answer 83

Simplified version of southern, norther, or western. Sample is spotted directly on a membrane, without electrophoretic separation. No information on size, but answers presence and quantity.

Question 84

what is in situ hybridization

Answer 84

Detects mRNA transcripts in whole tissue or tissue sections. Identifies where genes are expressed

Question 85

what is colony/plaque screening

Answer 85

Bacterial colonies or phage plaques are grown from library and replicated on membrane. Membrane is probed just like other blots and positive clones are identified and isolated

Question 86

what is a Microarray

Answer 86

similar to dot blot, but probes are immobilized on the substrate and the sample is labeled! Many genes can be analyzed at once, only limited by the number of probes. Can be spotted by machine so thousands can fit on microscope slide. After hybridization, the array is scanned and the intensity at each spot represents level of expression

Question 87

what is EMSA

Answer 87

Used to determine whether a protein binds to a candidate DNA sequence. DNA is labeled (must be short sequence) and incubated with protein, then run on polyacrylamide gel. DNA is then transferred to membrane and detected (blotting). Mobility of the probe will be slowed if bound to protein. Adding an antibody against the suspected protein reduces mobility further and confirms identity of the bound protein (Supershift)
see slide 32 for example