Chapter 7

Question 0

For gel electrophoresis, ____ has high resolving capability (distinguish DNA with size difference as little as a single base), while ___ has less resolving capability, but it can separate DNA molecules of up to 10 or even hundreds of kilobases.

Answer 0

Polyacrylamide has high resolving capability (distinguish DNA with size difference as little as a single base), while Agarose has less resolving capability, but it can separate DNA molecules of up to 10 or even hundreds of kilobases.

Question 1

Very long DNAs are unable to ___, even in agarose. DNA molecules above a certain size (30 to 50kb) cannot be resolved this way.
However, these very long DNAs can be resolved from one another, if the electric field is applied in pulses that are ___.This technique is known as ____. Electrodes are switched on and off alternatively, and DNA can snake its way through the gel.

Answer 1

Very long DNAs are unable to penetrate the pores even in agarose. DNA molecules above a certain size (30 to 50kb) cannot be resolved this way.
However, these very long DNAs can be resolved from one another, if the electric field is applied in pulses that are orientated orthogonally to each other. This technique is known as pulsed-field electrophoresis.
A and B represents two sets of electrodes. They are switched on and off alternatively, and DNA can snake its way through the gel.

Question 2

Electrophoresis separates DNA molecules not only according to their molecular weight, but also according to their ___ and ___.
• A circular DNA that is __ or ___ migrates more slowly than does a linear molecules of equal size. ____ DNAs, which are compact and have a small effective volume, migrate more rapidly during electrophoresis than relaxed circular DNA of equal mass.

Answer 2

Electrophoresis separates DNA molecules not only according to their molecular weight, but also according to their shape and topological properties.
• A circular DNA that is relaxed or nicked migrates more slowly than does a linear molecules of equal size. Supercoiled DNAs, which are compact and have a small effective volume, migrate more rapidly during electrophoresis than relaxed circular DNA of equal mass.

Question 3

Electrophoresis can also be used to separate RNAs as well. RNAs also have a uniform negative charge. But RNAs have extensive ___. To avoid those, RNAs can be treated with ___ that forms adducts with amino groups in the bases which prevents ___

Answer 3

Electrophoresis can also be used to separate RNAs as well. RNAs also have a uniform negative charge. But RNAs have extensive secondary and tertiary structures. To avoid that, RNAs can be treated with glyoxal that forms adducts with amino groups in the bases and prevent base pairing in RNAs)

Question 4

Restriction enzymes typically recognize short (4-8bp) target sequences, usually palindromic, and cut at a defined position within the sequences.
• One common used EcoR I (so named because it was found in certain strands of E. coli) recognizes and cleaves sequence 5’-GAATTC-3’.
• Some generates blunt ends, some generates sticky ends.
• If two enzymes generate the same ____, you can easily ligate the products from __ digestions.

Answer 4

Restriction enzymes typically recognize short (4-8bp) target sequences, usually palindromic, and cut at a defined position within the sequences.
• One common used EcoR I (so named because it was found in certain strands of E. coli) recognizes and cleaves sequence 5’-GAATTC-3’.
• Some generates blunt ends, some generates sticky ends.
• If two enzymes generate the same sticky ends, you can easily ligate the products from two digestions.

Question 5

Hybridization: ____.
• DNA hybridization can be used to detect ____ within complicated mixtures of nucleic acids.
• Probes can be a purified DNA fragment or a chemically synthesized DNA molecule. Probes can be end labeled using polynucleotide kinase in order to___ or ____. The labeled precursors are most commonly nucleotides modified with either a fluorescent dye or radioactive atoms.

Answer 5

Hybridization: complementary single-stranded polynucleotides can base pair to form a hybrid molecule.
• DNA hybridization can be used to detect specific sequences within complicated mixtures of nucleic acids.
• Probes can be purified DNA fragment or chemically synthesized DNA molecule. Probes can be end labeled using polynucleotide kinase to add γ-phosphate from ATP to the 5’-OH group of DNA, or to add labeled precursors when synthesizing new DNAs. The labeled precursors are most commonly nucleotides modified with either a fluorescent dye or radioactive atoms.

Question 6

DNA labeled with fluorescent precursors can be detected by illuminating the DNA sample with appropriate wavelength UV light and monitoring the longer-wavelength light that is emitted in response.
• Radioactively labeled precursors typically have radioactive 32P incorporated into the ____ in one of the four nucleotides. Radioactive DNA can be detected by ___or by photomultipliers that emit light in response to ____.

Answer 6

DNA labeled with fluorescent precursors can be detected by illuminating the DNA sample with appropriate wavelength UV light and monitoring the longer-wavelength light that is emitted in response.
• Radioactively labeled precursors typically have radioactive 32P incorporated into the α-phosphate in one of the four nucleotides. Radioactive DNA can be detected by exposing the sample of interest to X-ray film or by photomultipliers that emit light in response to excitation by the β particles emitted from 32P.

Question 7

Southern Blot hybridization: steps A-H

Answer 7

A. Cut DNA separated by electrophoresis;
B. The gel is soaked in alkli to denature DNA fragments;
C. DNA is transferred from the gel to positively charged membrane;
D. UV cross-link to fix the binding of DNA on membrane;
E. Probe DNA labeling;
F. Probe DNA hybridizes to the complementary DNA present on the membrane;
G. Low and high stringency washes to remove nonspecific binding;
H. X-ray film to detect the existence of the specific DNA in the mix.

Question 8

Northern blot can be used to identify ___ in a population of RNAs. mRNAs are relatively short (

Answer 8

Northern blot: can be used to identify a particular mRNA in a population of RNAs. mRNAs are relatively short (

Question 9

The availability of complete sequence information has enabled development of the “____” experiments. Thousands of ____ can be attached to a solid surface, typically a glass or plastic slide. Then, ____ generated from total RNAs from different tissues can be used to hybridize the array of probe DNAs. The intensity of hybridization in the array is a measure of the ____.

Answer 9

The availability of complete sequence information has enabled development of the “reverse hybridization” experiments. Thousands of known DNA can be attached to a solid surface, typically a glass or plastic slide. Labeled cDNA generated from total RNAs from different tissues (one with red label and one with green label) can be used to hybridize the array of probe DNAs. The intensity of hybridization in the array is a measure of the expression level of multiple genes.

Question 10

If you need a large amount of single pure DNA, or you need to make fusion protein to study gene function, you need DNA cloning.
• DNA cloning is to construct ___ and maintain them in cells. If you _____, you make a DNA library.

Answer 10

If you need a large amount of single pure DNA, or you need to make fusion protein to study gene function, you need DNA cloning.
• DNA cloning is to construct recombinant DNA molecules and maintain them in cells. If you clone a collection of DNAs into a given vector, you make a DNA library.

Question 11

DNA Cloning
The vector: contains ___; selection marker; has unique sites for one or more ___.
• Many vectors are small (~3kb) circular DNA molecules called plasmids. These molecules were originally derived from ___that are found in many bacteria an single cell eukaryotes. They can ___ independently of their host, and carry selectable markers.
• Some vectors have ___ that can drive the ___ within the insert DNA. They are called expression vectors.
• Vector DNA can be introduced into host organism by ____. You need competent cells. E. coli can be rendered competent for DNA uptake by treatment with ___ ions.

Answer 11

Vector: contains origin of replication; selection marker; has unique sites for one or more restriction enzymes.
• Many vectors are small (~3kb) circular DNA molecules called plasmids. These molecules were originally derived from extrachromosomal circular DNA molecules that are found in many bacteria an single cell eukaryotes. They can propagate independently of their host, and carry a selectable markers.
• Some vectors have transcriptional promoters that can drive the expression of genes within the insert DNA. They are called expression vectors.
• Vector DNA can be introduced into host organism by transformation. You need competent cells. E. coli can be rendered competent for DNA uptake by treatment with calcium ions.

Question 12

A DNA library is a population of identical vectors that each contains a different ____.
Genomic library: is most useful for generating DNA for ___.
cDNA library: make complementary DNA from ___, and construct library from the cDNA products.
Each transformed cells contains only ____. The colonies from cells carrying any cloned sequence of interest can be identified and the DNA retrieved.

Answer 12

A DNA library is a population of identical vectors that each contains a different DNA insert.
Construction and probing of a DNA library (right)
Genomic library: is most useful for generating DNA for sequencing a genome.
cDNA library: make complementary DNA from mRNAs, and construct library from the cDNA products.
Each transformed cells contains only a single vector with its associated insert DNA. The colonies from cells carrying any cloned sequence of interest can be identified and the DNA retrieved.

Question 13

Construction of a cDNA library:
3 steps

Answer 13

Construction of a cDNA library
1. RNA-dependent DNA polymerase reverse transcriptase (RT) transcribes RNA into cDNA (use oligoT as primer), generating mRNA-DNA duplex;
2. Remove RNA by treatment with NaOH, and the remaining single strand is used as template for second strand synthesis;
3. Use random 6bp primer (random hexamers) and DNA polymerase to generate double-stranded DNA products that can be cloned into a plasmid vector.

Question 14

Chemical synthesis of defined DNA sequences:
Oligonucleotides are custom-designed segments of ___.
Right now chemical synthesis of DNA molecules ___-___ bases long is efficient and accurate.
The precursors for nucleotide addition are chemically protected molecules called ____. In contrast to the direction of chain growth used by DNA polymerase, growth of the DNA chain is by addition to the __’ end of the molecule.

Answer 14

Chemical synthesis of defined DNA sequences
Oligonucleotides: custom-designed segments of single-stranded DNA.
Right now chemical synthesis of DNA molecules 10-100 bases long is efficient and accurate.
The precursors for nucleotide addition are chemically protected molecules called phosphoamidines. In contrast to the direction of chain growth used by DNA polymerase, growth of the DNA chain is by addition to the 5’ end of the molecule.

Question 15

A custom-designed oligonucleotide carrying a mismatch to a segment of cloned DNA can be used to create ____. The method is called site- directed mutagenesis, which is performed as follows: ____

Answer 15

Custom-designed oligonucleotide carrying a mismatch to a segment of cloned DNA can be used to create a directed mutation in that cloned DNA. The method is called site- directed mutagenesis, which is performed as follows: the oligonucleotide is hybridized to the cloned DNA fragment and used to prime DNA synthesis with cloned DNA as template.

Question 16

The Polymerase Chain Reaction (PCR) amplifies DNA by _____.
This game-changing method from amplifying particular segments of DNA, distinct from cloning and propagation with a host cell, is called polymerase chain reaction (PCR).
PCR uses ____ to add nucleotides to the 3’ end of a custom-designed oligonucleotide when it is annealed to the long template DNA.

Answer 16

The Polymerase Chain Reaction (PCR) amplifies DNA by repeated rounds of DNA replication in vitro

PCR uses DNA polymerase to add nucleotides to the 3’ end of a custom-designed oligonucleotide when it is annealed to the long template DNA.

Question 17

PCR and Forensics:
A suspect’s DNA contains a polymorphism that is presents in the DNA found at the scene of the crime, which can be determined by PCR and sequencing.
• Polymorphisms are ____ found in a population of organisms at a common, homologous region of the chromosome, such as a gene. A polymorphism can be as simple as ____ among different members of the population, or a difference in the ___, such as CA.
• We can amplify the DNA surrounding and including the site of polymorphism and subject it to sequencing. The nucleotide sequence helps to determine whether two DNA samples match.

Answer 17

A suspect’s DNA contains a polymorphism that is presents in DNA found at the scene of the crime, which can be determined by PCR and sequencing.
• Polymorphisms are alternative DNA sequences (alleles) found in a population of organisms at a common, homologous region of the chromosome, such as a gene. A polymorphism can be as simple as alternative, single-base difference as the same site in the chromosome among different members of the population, or difference in the length of a simple nucleotide repeat sequence such as CA.
• We can amplify the DNA surrounding and including the site of polymorphism and subject it to sequencing. The nucleotide sequence helps to determine whether two DNA samples match.

Question 18

A major technical advance in DNA sequencing came from the use of
fluorescent chain-terminating nucleotides. The chains can be separated by ___. In this way, a single column produces 600-800bp of DNA sequence for less than 3 h of size separation.
Automatic ______ (sequenators) were designed to have 384 separate fraction columns. In principle this can generate 200kb of raw DNA sequence in just a few hours.
A cluster of ___ such machines can generate the equivalent of human genome Genome, 3X 10~9bp, in just two months.
Next-generation sequenators can routinely produce the equivalent of a complete human genome is a single run of just a few hours.

Answer 18

A major technical advance in DNA sequencing came from the use of
fluorescent chain-terminating nucleotides. The chains can be separated by column chromography. In this way, a single column produces 600-800bp of DNA sequence for less than 3 h of size separation.
Automatic sequencing machines (sequenators) were designed to have 384 separate fraction columns. In principle this can generate 200kb of raw DNA sequence in just a few hours.
A cluster of 100 such machines can generate the equivalent of human genome Genome, 3X 10~9bp, in just two months.
Next-generation sequenators can routinely produce the equivalent of a complete human genome is a single run of just a few hours.

Question 19

Shotgun Sequencing
• Multiple copies of the genome are randomly shredded into pieces by squeezing the DNA through a _____. This is done a second time to generate pieces that are 10,000 bp long
• Each 2,000 and 10,000 bp fragment is inserted into a plasmid
– The two collections of plasmids containing 2,000 and 10,000 bp chunks of DNA are plasmid libraries
• Both the 2,000 and the 10,000 bp plasmid libraries are sequenced. 500 bp from each end of each fragment are decoded generating millions of sequences Sequencing both ends of each insert is critical for _____. (10X sequencing coverage)
• Computer algorithms assemble the millions of sequenced fragments into a continuous stretch resembling each ___

Answer 19

Shotgun Sequencing
• Multiple copies of the genome are randomly shredded into pieces by squeezing the DNA through a pressurized syringe. This is done a second time to generate pieces that are 10,000 bp long
• Each 2,000 and 10,000 bp fragment is inserted into a plasmid
– The two collections of plasmids containing 2,000 and 10,000 bp chunks of DNA are plasmid libraries
• Both the 2,000 and the 10,000 bp plasmid libraries are sequenced. 500 bp from each end of each fragment are decoded generating millions of sequences Sequencing both ends of each insert is critical for the assembling the entire chromosome. (10X sequencing coverage)
• Computer algorithms assemble the millions of sequenced fragments into a continuous stretch resembling each chromosome

Question 20

Shotgun strategy permits a partial assembly of large genome sequences
• The average human chromosome is composed of __ Mb. The rate-limiting step in determining their complete sequence is ___, rather than the production of the data per se.

Answer 20

Shotgun strategy permits a partial assembly of large genome sequences
• The average human chromosome is composed of 150 Mb. The rate-limiting step in determining their complete sequence is data analysis, rather than the production of the data per se.

Question 21

Contigs are linked by sequencing the ends of large DNA fragments
• ___ are large contiguous sequences. The use of end sequences derived from the larger fragments carried in the 5-kb and 100-kb insert clones can facilitate the assembly of the genome.

Answer 21

Contigs are linked by sequencing the ends of large DNA fragments
• Contigs: large contiguous sequences. The use of end sequences derived from the larger fragments carried in the 5-kb and 100-kb insert clones can facilitate the assembly of the genome.

Question 22

The paired-end strategy permits the assembly of large-genome scaffolds
• A major limitation to producing larger contigs is the occurrence of ___. Such sequences complicate the assembly process. One method that is used to overcome this difficulty is called paired-end sequencing.

Paired end sequencing:
• Generate library of 5-kb fragments, and sequence both ends. Because the repetitive sequences are less than 2-3 kb in length, the paired-end sequences for the 5-kb insert are sufficient to span contigs interrupted by repetitive DNAs.
• To create paired-end sequence data from large DNA fragments that are larger than 100kn in length, you need to use a special _____. BAC permits the assignment of multiple contigs into a single scalfold of several megabases.

Answer 22

The paired-end strategy permits the assembly of large- genome scaffolds
• A major limitation to producing larger contigs is the occurrence of repetitive DNAs. Such sequences complicate the assembly process. One method that is used to overcome this difficulty is called paired-end sequencing.
• Generate library of 5-kb fragments, and sequence both ends. Because the repetitive sequences are less than 2-3 kb in length, the paired-end sequences for the 5-kb insert are sufficient to span contigs interrupted by repetitive DNAs.
• To create paired-end sequence data from large DNA fragments that are larger than 100kn in length, you need to use a special cloning vector called bacterial artificial chromosomes (BAC) (150kbp to 350kbp in size). BAC permits the assignment of multiple contigs into a single scalfold of several megabases.

Question 23

Card 1. Nanotechnology to produce rapid and inexpensive genome sequencing.
• genomic DNA is isolated, fragmented, ligated to adapters and separated into single strands.
• Small fragments of DNA is mixed with small beads (DNA is diluted so that one DNA binds to 1 single bead).
• The DNA-containing beads are dispersed on a silicon plate consisting of 400,000 regularly spaced picoliter-sized wells (each well captures a bead)
• The beads are captured in the droplets of a __ and PCR amplification occurs within each droplet. PCR is performed directly on the bead-tethered DNAs to amplify each DNA molecules.
• The __ ___ of DNA molecules is created in each well, which is then used as a template for an additional round of DNA synthesis.

Answer 23

Nanotechnology to produce rapid and inexpensive genome sequencing.
• genomic DNA is isolated, fragmented, ligated to adapters and separated into single strands.
• Small fragments of DNA is mixed with small beads (DNA is diluted so that one DNA binds to 1 single bead).
• The DNA-containing beads are dispersed on a silicon plate consisting of 400,000 regularly spaced picoliter- sized wells (each well captures a bead)
• The beads are captured in the droplets of a PCR-reaction-mixture-in-oil emulsion and PCR amplification occurs within each droplet. PCR is performed directly on the beads-tethered DNAs to amplify each DNA molecules.
• The homogenous population of DNA molecules is created in each well, which is then used as a template for an additional round of DNA synthesis.

Question 24

Card 2. Nanotechnology to produce rapid and inexpensive
Sequencing is performed in stepwise fashion with the plate being separately exposed to dATP, dGTP, dCTP and dTTP sequentially, with a washing cycle between each pulse of dNTP substrate.
• The incorporation of a deoxynucleotide depends on the ____and the ____
• This reaction promotes a enzymatic reaction that produces ___, which are detected by a microprocessor attached to a computer.
• The light pulses indicate which nucleotide is incorporated in each well, thereby producing the sequence of the DNA contained in all 400,000 wells.

Answer 24

Sequencing is performed in stepwise fashion with the plate being separately exposed to dATP, dGTP, dCTP and dTTP sequentially, with a washing cycle between each pulse of dNTP substrate.
• The incorporation of a deoxynucleotide depends on the presence of the complementary base in the template and results in the liberation of pyrophosphate.
• This reaction promotes a enzymatic reaction that produces pulses of light, which are detected by a microprocessor attached to a computer.
• The light pulses indicate which nucleotide is incorporated in each well, thereby producing the sequence of the DNA contained in all 400,000 wells.

Question 25

Whole-genome tiling assays are use to visualize the transcriptome
• Transcriptome: The portion of an organism’s genome that ____.
• Whole-genome tiling array: __ are spotted on a glass or silicon slide. Typically one oligonucleotide is produced for every 100-150 bp of DNA sequence in a sequential manner across the genome, resulting a tiling array of DNA sequences. Right now it is feasible to produce a complete arrays on a single glass slide or silicon chip that is just 1cm~2 in size.
• To visualize the transcriptome, the tiling arrays are ______. An alternative strategy for transcriptome profiling is the ___ of cDNAs prepared from cells or tissues.

Answer 25

Whole-genome tiling assays are use to visualize the transcriptome
• Transcriptome: The portion of an organism’s genome that acts as a template for RNA synthesis is known as the transcriptome.
• Whole-genome tiling array: synthetic single- stranded DNAs of 50 nucs are spotted on a glass or silicon slide. Typically one oligonucleotide is produced for every 100-150 bp of DNA sequence in a sequential manner across the genome, resulting a tiling array of DNA sequences. Right now it is feasible to produce a complete arrays on a single glass slide or silicon chip that is just 1cm~2 in size.
• To visualize the transcriptome, the tiling arrays are hybridized with fluorescently labeled RNA or cDNAs. An alternative strategy for transcriptome profiling is the high-throughput sequencing of cDNAs prepared from cells or tissues.

Question 26

Whole genome tiling array reveals details of __-__ structure of a gene.
Intronic transcripts are ___, So the exonic sequences display more intense signals than introns.
Another useful feature of whole-genome tiling arrays is that they detect ___, such as microRNAs.
The ____ transcript units are easily detected by hybridization to tiling arrays. In some cases, miRNA genes contain Introns that must be ___ before transcripts are also detected.

Answer 26

Whole genome tiling array reveals details of intron- exon structure of a gene.
Intronic transcripts are unstable. So the exonic sequences display more intense signals than introns.
White arrow shows a large intronic region that might contain a small exon. Another useful feature of whole-genome tiling arrays is that they detect non- coding sequences, such as microRNAs. The pri-RNA transcript units are easily detected by hybridization to tiling arrays. In some cases, miRNA genes contain Introns that must be processed before transcripts are also detected.

Question 27

Regulatory DNA sequences can be identified by using specialized alignment tools
• BLAST searches permit the rapid comparison and alignment of either ____or___.
• A computer program called VISTA aligns ____, on the order of 10-20bp, and thereby identifies imperfectly conserved non-coding sequences over a stretch of just 50-75bp. It is conceivable that many of them correspond to ____ enhancers.

Answer 27

Regulatory DNA sequences can be identified by using specialized alignment tools
• BLAST searches permit the rapid comparison and alignment of either protein or nucleic acids sequences.
• A computer program called VISTA aligns the sequences contained in genomes of different related organisms over short windows, on the order of 10-20bp, and thereby identifies imperfectly conserved non-coding sequences over a stretch of just 50-75bp. It is conceivable that many of them correspond to tissue-specific enhancers.

Question 28

The purification of individual proteins is critical to understanding their function.
• Each protein has unique properties that makes its purification somewhat different. This is in contrast to different DNAs, which all share the same helical structure and are only distinguished by their precise sequence.
• The purification of a protein is designed to exploit its unique characteristics, including size, charge, shape, and in many instances, function.
• Purification of a protein requires a specific assay. For DNA purification, we can use __ to ____. For protein, we can use an ___ to detect specific proteins in the same way.
In many instances, it is more convenient to use a more direct measure for the function of the protein. For example, a specific DNA-binding protein can be assayed by determining ___. (Electrophoretic mobility shift assay).
• For preparing cell extract, the cells can be lysed by __, ___, treatment with ___, or rapid change in ___. The goal is to weaken and break the membrane surrounding the cells to allow proteins to escape. Some times you can freeze the cells before applying shearing forces.

Answer 28

The purification of individual proteins is critical to understanding their function.
• Each protein has unique properties that makes its purification somewhat different. This is in contrast to different DNAs, which all share the same helical structure and are only distinguished by their precise sequence.
• The purification of a proteins is designed to exploit its unique characteristics, including size, charge, shape, and in many instances, function.
• Purification of a protein requires a specific assay. For DNA purification, we can use hybridization to bind the DNA to its complement. For protein, we can use an anti- body to detect specific proteins in the same way. In many instances, it is more convenient to use a more direct measure for the function of the protein. For example, a specific DNA-binding protein can be assayed by determining its interaction with the appropriate DNA. (Electrophoretic mobility shift assay).
• For preparing cell extract, the cells can be lysed by detergent, shearing forces, treatment with low ionic salt, or rapid change in pressure. The goal is to weaken and break the membrane surrounding the cells to allow proteins to escape. Some times you can freeze the cells before applying shearing forces.

Question 29

Proteins can be separated from one another using column chromatography: What are the 2 kinds and how do they work?

In each case, a glass tube is filled with beads, and the protein mixture is passed through the matrix. The nature of beads dictates the basis for protein separation.

Answer 29

Proteins can be separated from one another using column chromatography
1. Ion-exchangechromatography 2. Gel-filtrationchromatography.

In 1, the beads are negatively charged, so the positive charged proteins binds and are retained on the column, whereas the negative charged proteins pass through. Proteins that interact more strongly need more salt to be eluted
In 2, the beads contain aqueous spaces into which small proteins can pass, slowing down their progress through the column,. Larger protein can not enter the beads, allowing them to pass more rapidly through the column.

Question 30

Affinity chromatography
Specific DNA sequence or ATP can be coupled to beads, which can be used to purify proteins that bind to ATP or the specific DNA.
After binding and washing, increased concentration of ATP or DNA oligo will elute the protein according to their affinity.

One common form of protein affinity chromatography is ___ chromatography. How does this approach work?

Answer 30

Affinity chromatography
Specific DNA sequence or ATP can be coupled to beads, which can be used to purify proteins that bind to ATP or the specific DNA.
After binding and washing, increased concentration of ATP or DNA oligo will elute the protein according to their affinity.
One common form of protein affinity chromatography is immunoaffinity chromatography. In this approach , an antibody that is specific for the target protein is attached to the beads. The antibody will interact only with the target protein, and the bound protein can be eluted from the column using salt, PH gradient, or in some cases, mild detergent.

Question 31

Protein purification using peptide tag
Epitopes (a sequence of 7- 10 amino acids recognized by an antibody).
Immunoprecipitation can be used to rapidly ___.
Co-immunoprecipitation (Co-IP), or ChIP (chromatin-IP) can be a useful method to determine ___

Answer 31

Protein purification using peptide tag
Epitopes ( a sequence of 7- 10 amino acids recognized by an antibody).
Immunoprecipitation can be used rapidly purify protein or protein complexes from crude extract.
Co-immunoprecipitation (Co-IP), or ChIP (chromatin-IP) can be useful method to determine what protein or other molecules are associated with the target protein

Question 32

Separation of protein on polyacrylamide gels:
SDS gel electrophoresis: addition of strong ionic detergent SDS (sodium dodecyl sulfate) and ___ denatures the proteins and provides each with a uniform ___ charge. Separation on the basis of size is achieved by electrophoresis.

Immunoblotting: After separation by electrophoresis, proteins are transferred to filter paper in a manner that retain the same relative position of the proteins. After blocking _____, an antibody to the protein of interest is added to the filter paper. The site of antibody binding is then detected using an attached enzyme that creates light when it ____.

Answer 32

Separation of protein on polyacrylamide gels
SDS gel electrophoresis: addition of strong ionic detergent SDS (sodium dodecyl sulfate) and β- mercaptoethanol denatures the proteins and provides each with uniform negative charge. Separation on the basis of size is achieved by electrophoresis.
Immunoblotting: After separation by electrophoresis, proteins are transferred to filter paper in a manner that retain the same relative position of the proteins. After blocking nonspecific proteins-binding sites, antibody to the protein of interest is added to the filter paper. The site of antibody binding is then detected using an attached enzyme that creates light when it acts on the substrate.

Question 33

Protein molecules can be directly sequenced:
Although more complex than the sequencing of nucleic acids, protein molecules can also be sequenced. Two widely used methods for determining protein sequence are ___ using an automated protein sequencer and ____.
Edman degradation: is a chemical reaction in which the amino acid’ residues are _____. The free α-amino group can be modified by PITC. The derived amino acid is then cleaved off the polypeptide by treatment with __ under conditions that don’t destroy the remaining peptide bonds. The identity of the released amino acid derivative can be determined by its __ profile using a column chromatography method called ____.

Answer 33

Protein molecules can be directly sequenced
Although more complex than the sequencing of nucleic acids, protein molecules can also be sequenced. Two widely used methods for determining protein sequence are Edman degradation using an automated protein sequencer and tandem mass spectrometry.
Edman degradation: is a chemical reaction in which the amino acid’ residues are sequentially released from the amino terminus of a polypeptide chain. The free α- amino group can be modified by PITC. The derived amino acid is then cleaved off the polypeptide by treatment with acid under conditions that don’t destroy the remaining peptide bonds. The identity of the released amino acid derivative can be determined by its elution profile using a column chromatography method called high- performance liquid chromatography (HPLC)

Question 34

Tandem Mass Spectrometry (MS/MS):
Tandem mass spectrometry (MS/MS) is the most common method in use today to determine __. The principle is material travels through a instrument (in a vacuum) in a manner that is sensitive to its ___ ratio.
To use MS/MS, the protein of interest is usually digested into short peptides (less than 20 amino acids) by digestion with specific ___ such as trypsin. The mixture of peptides is subjected to __, and each individual peptide will be separated from the others in a mixture by its mass/charge ratio. The individual peptides are then captured and fragmented into all of the component peptides (collision-induced dissociation), and the mass of each of these component fragments is then determined.

Answer 34

Tandem Mass
Spectrometry (MS/MS)
Tandem mass spectrometry (MS/MS) is the most common method in use today to determine protein sequence. The principle is material travels through a instrument (in a vacuum) in a manner that is sensitive to its mass/charge ratio.
To use MS/MS, the protein of interest is usually digested into short peptides (less than 20 amino acids) by digestion with specific protease such as trypsin. The mixture of peptides is subjected to mass spectrometry, and each individual peptide will be separated from the others in a mixture by its mass/charge ratio. The individual peptides are then captured and fragmented into all of the component peptides (collision-induced dissociation), and the mass of each of these component fragments is then determined.

Question 35

The goal of proteomics is the identification of the full set of ___ under a particular set of conditions (Called proteome), their relative abundance, their modifications and their interaction partner proteins.
• Combined ___with ___identifies individual proteins within a complex extract: protein digestion with ___; fractionation by ion exchange chromatography (based on ionic interactions) and reverse phase column (based on hydrophobic interaction with the column material); this generates ____that can be distinguished from each other and sequenced.
• In practice, this methods detects only a subsets of proteins in a complex mixture of proteins. A typical analysis can detect approximately 1000 different proteins. It is difficult to determine the abundance of protein by this approach.

Answer 35

The goal of proteomics is the identification of the full set of proteins produced by a cell or tissue under a particular set of conditions (Called proteome), their relative abundance, their modifications and their interaction partner proteins.
• Combined liquid chromatography with mass spectrometry identifies individual proteins within a complex extract: protein digestion with trypsin; fractionation by ion exchange chromatography (based on ionic interactions) and reverse phase column (based on hydrophobic interaction with the column material); this generates many lower-complexity mixture of peptides can be distinguished from each other and sequenced.
• In practice, this methods detects only a subsets of proteins in a complex mixture of proteins. A typical analysis can detect approximately 1000 different proteins. It is difficult to determine the abundance of protein by this approach.

Question 36

Mass spectrometry can also monitor protein modification states:
• Protein modification can profoundly affect its function. Phosphorylation can cause a protein to __ in a functionally important manner. Other modifications include: ___, __, and ___ (add the 76-amino-acid ubiquitin to a lysine residue via a pseudopeptide bond, target for protein degradation).
• Phosphoproteome: the complete set of phosphorylated proteins in the cell.
Affinity resins that include immobilized ____ can specifically bind phosphorylated peptides. Mixtures of peptides derived from crude cell extracts can be incubated with such a resin, and the small proportion of peptides that bind are enriched for phosphopeptides. These peptides can be analyzed using ___ to identify the proteins that are modified and the sites of modification.

Answer 36

Mass spectrometry can also monitor protein modification states
• Protein modification can profoundly affect its function. Phosphorylation can cause a protein to alter its conformation in a functionally important manner. Other modifications include: methylation, acetylation, and ubiquitylation (add the 76-amino-acid ubiquitin to a lysine residue via a pseudopeptide bond, target for protein degradation).
• Phosphoproteome: the complete set of phosphorylated proteins in the cell.
Affinity resins that include immobilized Fe+++ can specifically bind phosphorylated peptides. Mixtures of peptides derived from crude cell extracts can be incubated with such a resin, and the small proportion of peptides that bind are enriched for phosphopeptides. These peptides can be analyzed using LC-MS to identify the proteins that are modified and the sites of modification.

Question 37

Protein-protein interactions can yield information regarding protein function:
• Proteomics is also concerned with identifying all of the proteins that associate with another protein in a cell to generate ___. Interactome can be used to determine ____. Proteins that are part of the same protein complex will frequently be involved in the same cellular process.
• One method for determining protein-protein interaction is the _____. A second approach is to use ____ or immunoprecipitation to rapidly purify a protein of interest along with any associated proteins. The resulting mixed proteins can be analyzed by LC-MS to identify associated proteins.

Answer 37

Protein-protein interactions can yield information regarding protein function
• Proteomics is also concerned with identifying all of the proteins that associate with another protein in a cell to generate interactome. Interactome can be used to determine which processes a protein may be involved. Proteins that are part of the same protein complex will frequently be involved in the same cellular process.
• One method for determine protein-protein interaction is the yeast two-hybrid assay. A second approach is to use affinity resins or immunoprecipitation to rapidly purify a protein of interest along with any associated proteins. The resulting mixed proteins can be analyzed by LC-MS to identify associated proteins.

Question 38

DNA-bound protein protects the DNA from nuclease and chemical modification
DNA footprinting principle: if a DNA fragment is labeled with a radioactive atom only at the end of one strand, then the __ can be labeled from the size of the labeled fragment that results.

Answer 38

DNA footprinting principle: if a DNA fragment is labeled with radioactive atom only at the end of one strand, then the location of any break in the strand can be labeled from the size of the labeled fragment that results.

Question 39

1. Nuclease protection footprinting: ? (The resulting “footprint” is revealed by the absence of bands of sizes that correspond to the site of protein binding.
2. Chemical protection footprinting: relies on the ability of a bound protein to ?
3. Chemical interference footprinting: Determines what? (EMSA with modified DNA). Chemical modification example: ethylynitrosourea (ENU) modification of the phosphate residues in the backbone of DNA.
   For DNA footprinting to be effective, >___% of the DNA must be bound by the protein, because we need to detect the absence of a signal. EMSA is more sensitive.

Answer 39

1. Nuclease protection footprinting: DNA molecules are cut at random by DNase unless they are protected by a protein. (The resulting “footprint” is revealed by the absence of bands of sizes that correspond to the site of protein binding.
2. Chemical protection footprinting: relies on the ability of a bound protein to protect bases in the binding site from base- specific chemical reagent that give rise to a backbone cuts.
3. Chemical interference footprinting: determines which features of the DNA structure is necessary for the protein to bind. (EMSA with modified DNA). Chemical modification example: ethylynitrosourea (ENU) modification of the phosphate residues in the backbone of DNA.
   For DNA footprinting to be effective, >90% of the DNA must be bound by the protein, because we need to detect the absence of a signal. EMSA is more sensitive.

Question 40

Chromatin immunoprecipitation (ChIP) can detect ____ in the cell
• ChIP is a powerful technique to monitor protein-nucleic acid interactions in cells. What are its 4 steps?

Answer 40

Chromatin immunoprecipitation (ChIP) can detect protein association with DNA in the cell

ChIP:
A. Use formaldehyde to cross-link DNA to any bound proteins;
B. Lyse cell and break DNA into small pieces (200-300bp) by sonication;
C. Use an specific antibody to immunoprecipitate DNA attached to the target protein.
D. Reverse the cross-link between DNA and protein, analyze the DNA by PCR or sequencing.a

Question 41

ChIP-chip: DNAs from two ChIPs (under two different conditions) are labeled with two different fluorophores, and hybridized to
tiling DNA microarrays.
ChIP-chip limitations: describe two

Answer 41

ChIP-chip limitations:
1. Resolution: can only demonstrate the protein bind to a 200-300bp fragment, not more short specific sequence;
2. Antibody:proteinscanonlybe identified only if the relevant epitope (sequence that is recognized by antibody) is exposed when the protein is cross-linked to the DNA

Question 42

ChIP-seq: ____ DNA is subjected to direct DNA sequencing.
• Easier than ChlP-chip , but you need to know ____ of the organism to map the sites of DNA binding.

Answer 42

ChIP-seq: immunoprecipitated DNA is subjected to direct DNA sequencing.

Easier, but you need to know the genomic sequence of the organism to map the sites of DNA binding.

Question 43

Chromosome conformation capture assays are used to analyze long-range interactions
• Chromosome conformation capture assays (3C): what are the 4 steps?

• 4C and 5C are variants of the 3C method that permit detection of _____ with a fixed anchor point within the genome. (Hoxd enhancers)

Answer 43

Chromosome conformation capture assays are used to analyze long-range interactions
• Chromosome conformation capture assays (3C): detect long-range interactions.
A. Use formaldehyde to cross-link protein to DNA;
B. The chromatin is broken up by digestion with restriction enzymes or sonication;
C. Ligate the resulting DNA;
D. Cross-linking is reversed, and the ligation mix is purified. Alternatively, the mix can be immunoprecipitated with a specific antibody.
• 4C and 5C are variants of the 3C method that permit detection of all chromosomal interactions with a fixed anchor point within the genome. (Hoxd enhancers)

Question 44

In vitro selection can be used to identify a protein’s ___.
• How can the DNA sequence that is recognized by a particular protein be identified?
• SELEX (systematic evolution of ligands by exponential enrichment). What are the 5 steps?

Answer 44

In vitro selection can be used to identify a protein’s DNA- or RNA-binding site
• How can the DNA sequence recognized by a particular protein be identified?
• SELEX (systematic evolution of ligands by exponential enrichment).
A. Produce a large library of ssDNA oligonucleotides using chemical DNA synthesis (the middle 10-12 bases of the oligo is randomized);
B. A short primer is annealed to the 3’ end of the oligo and extended to convert the randomized ssDNA library to a randomized dsDNA libaray;
C. DNAs enriched for binding a particular protein separated by EMSA or immunoprecipitation are then amplified;
D. Repeat the binding, enrichment and amplification steps to further enrich the sequence bound by the protein of interest;
E. DNA sequencing and computational analysis of the frequency of bases at each position.

Question 45

How is DNA visualized after gel electrophoresis?

Answer 45

Using a dye and UV light

Question 46

Restriction ending lease XhoI recognizes the sequence 5’-CTCGAG-3’ and cleaves bt the C and the T on each strand. What is the calculated frequency of this seq occurring in the genome?

Answer 46

This enzyme recognizes a 6 bp seqs, so the freq of finding it in a given genome is 4^6, or 1 in 4096 bps

Question 47

The restriction endonuclease SalI recognizes the seq 5’-GTCGAC-3’ and cleaves between the G and the T. Do you think sticky ends produced after XhoI and SalI cleavage could adhere to each other?

XhoI recognizes 5’-CTCGAG-3’, cleaving bt C and T.

Answer 47

Yes, even though the two enzymes has different recognition seqs, the sticky ends can bp with each other bc the single stranded regions are complimentary to each other.

Question 48

Describe two methods for labeling a DNA probe

Answer 48

1. Adding a label at the end of an intact DNA molecule
2. Labeling by incorporation: synthesizing new DNA in the presence of a labeled precursor. Done by performing PCR w a labeled precursor or by hybridizating the precursor with random hexameric oligonucleotides to DNA.

Question 49

Compare and contrast Southern blot and Northern blot

Answer 49

Southern blot: detecting a specific DNA seq with a DNA probe. You digest genomic DNA with a restriction enzyme, separate the DNA fragments via gel and transfer them to a q+ membrane and then detect a fragment of DNA that contains your DNA of interest with the probe.

Northern blot: detect a specific mRNA seq with a DNA probe. Similar steps as a Southern blot, except the mRNA is not digested. You can detect a certain amount of mRNA and compare it to another sample produced under diff experimental conditions.

Question 50

Describe to role of each feature of a plasmid:

1. Origin of rep
2. Restriction enzyme recognition sites
3. Selectable marker
4. Promoter

Answer 50

1. Origin of rep:
2. Restriction enzyme recognition sites:
3. Selectable marker:
4. Promoter:

Question 51

How does a genomic library differ from a cDNA library?
How are they similar?
What’s the advantage to using a cDNA library?

Answer 51

A genomic library consists of the complete set of DNA fragments, generated by restriction endonuclease digestion of the entire genome. A cDNA library consists of only expressed seqs in the genomic DNA. A cDNA is generated by reverse transcription of all cellular mRNA.
In each case, the resulting fragments are lighted into plasmid vectors

cDNA is favorable bc the genome consists of many noncoding DNA including seqs that encode for introns which get spliced out of the mRNA. cDNA libraries are useful for studying and expressing gene-encoding seqs.

Question 52

For PCR:
Why is the first step carried out at 94° C?
What happens in the rxn when the temp shifts to 55° C during cycling?
During cycling, what occurs at 72° C?

Answer 52

Why is the first step carried out at 94° C? To denature the DNA
What happens in the rxn when the temp shifts to 55° C during cycling?
During cycling, what occurs at 72° C?

Question 53

What is the basis for separation of proteins for:
Ion exchange
Gel-filtration
Affinity column chromatography

Answer 53

Ion exchange: separates proteins based on charge
Gel-filtration: separates based on size
Affinity column chromatography: separates based on their interaction with a specific molecule, protein, or nucleic acid that is coupled to the beads.

Question 54

What is the purpose of adding SDS to protein samples for Polyacrylamide gel electrophoresis?
What is mercaptoethanol used for?

Answer 54

SDS is used to eliminate any secondary, tertiary, and quaternary structures of proteins and coats all of its AAs with a uniform negative charge.

M is used to reduce disulfide bridges formed by cysteine residues

Question 55

Describe the electrophoretic mobility-shift array assay for testing the interactions between proteins and DNA.

Answer 55

1. Electrophoretic mobility-shift array: a protein is mixed withradiolabeled probe DNA that contains a binding site for that protein. Using acrylimide gel electrophoresis, there will be a band corresponding to free DNA & a band corresponding to the DNA in complex with the protein (a shorter band due to the added weight of the protein)

Question 56

In a DNA foot printing assay, explain why only one strand of the DNA can be end labeled in order for the experiment to work

Answer 56

Only one strand (end) is labeled so that nuclease digestion of the bound DNA fragment will produce, after gel electrophoresis, a visible ladder of fragments extending from a single, labeled end. Digestion of a strand labeled at both ends would complicate the pattern and obscure the “footprint “. And, if the protein binds asymmetrically, the pattern becomes even more complicated.

Question 57

Following the immunoprecipitation step in a chromatin immunoprecipitation (ChIP), explain how to identify the DNA seqs that remain bound to the protein of interest.

Answer 57

To determine if a specific known region is bound to the protein, use primers that are specific to those seqs in order to amplify that seq and compare the results to necessary controls. Another option is to use a tiling DNA microarray to identify many different seqs.

Question 58

______ can cause changes in protein recognition by an antibody on a Western blot. What can combat this issue if presented?

Answer 58

Alternative splicing of RNA

Use a new antibody that is polyclonal to the entire protein or monoclonal against a central or amino-terminal region of the protein.

Question 59

Describe the chain terminating nucleotides/dideoxy sequencing method. How does it work?

Answer 59

This method uses special modified substrates called ddNTPs which lack the OH on both of the ribose’s 2’ and 3’. DNA pol will incorporate the ddNTPs but once incorporated, synthesis will stop bc the lack of an OH on 3’ prevents addition of further nucs

Question 59

Describe the nuclease protection foot printing for testing the interactions between proteins and DNA.

Answer 59

DNA and end labeled DNA are incubated together and briefly exposed to a DNase. The sites bound by the protein are protected from nuclease cleavage, resulting in a region of DNA with cut sites. Shows as a gap on a gel

Question 60

Describe the chemical interference foot printing for testing the interactions between proteins and DNA.

Answer 60

This method determines which features of the DNA structure are necessary for the protein to bind. DNA is modified and incubated with the DNA binding protein, and protein-DNA complexes are isolated via EMSA. After detection of the labeled DNA in the gel, the shifted (bound by a protein) and unshifted (unbound) DNA can easily be separated. If the DNA modification made the protein unable to bind, there will be no modified DNA in the protein-DNA complex and vice versa.