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61 Cards in this Set

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Outline the structure of DNA (3 components).

Describe the assembly and structure of DNA.
Nucleotides linked by phosphodiester bonds, consisting of purine (AG) or pyrimidine (CT) base; deoxyribose sugar, and phosphate.

5'-3' antiparallel strands with H bonding b/w base pairs.

Helix is 20 bp/coil, 2 nm wide, 3.4 nm up, with major and minor grooves allowing for proteins to bind.
Define hybridization and denaturation.

What are the difference b/w high and low stringency conditions? When is each useful?
Hybridization = H-binding of 2 complimentary strands when cool.

Denaturation = breaking of H bonds b/w complimentary strands; occurs with heat.

High stringency conditions = hybridization only occurs between exactly complementary strands. Occurs at high T and low ionic strength.

Low-stringency: can allow hybridization of not exactly complementary strands. Useful for probe ID b/w species.
Describe the packing of DNA.
Double helix organized into nucleosomes by winding DNA around histone proteins.

Histones are organized into solenoids which are folded into chromosomes.
List the classifications of non-gene-coding DNA. (2)
Pseudogenes: similar to functional genes but transcriptionally or translationally inactive because they are riddled with mutations.

Repetitive DNA: stretches of DNA repeated 1000-1 000 000 x in genome. 40-48%.
What are the classifications of repetitive DNA?
Dispersed repetitive RNA: 2 types: SINES 90-500 bp and LINES 6000-7000 bp. E.g. Alu family (SINE) 700 bp repeat found by Alu RE. This copies self and inserts into other, possibly coding areas.

Satellite DNA: clustered in certain chromosomes. May be alpha-satellite DNA, minisatellites or microsatellites.

Alpha-satellite: tandem repeats of 171 bp for several million bp, found near centromere.

Minisatellite: 20-120 bp, in tandem to 1000 total.

Microsatellite: 2-5 bp. in tandem to 100 total.

Mini and micro used for gene mapping.
Why is mitochondrial DNA involved in many genetic dx?

Where is it inherited via?
Replicates with high mutation rate.

Maternally inherited from mitochondrion of fertilized egg.
How does RNA differ from DNA?

What is the role of RNA polymerase? Give the 4 types. What is it inhibited by?
Still nucleotides with phosphodiester linkages, but use ribose sugar and uracil instead of thymine. May be ss or ds, and can hybridize with cDNA.

RNA polymerase generates RNA. I = rRNA (large!). II = mRNA. III tRNA and 5SrRNA. Mitochondrial = transcribe mitochondrial genes.

Inhibited by alpha-amanitin - inhibits II and III at high concentrations. NOT I or IV.
What are eukaryotic genes composed of?

How does mature RNA differ from the transcription product?
Coding segments (genes) = exons; introns which contain the transcription start site (5' non-coding) and termination site (poly A addition site AATAAA 3')

Introns are removed, cap at 5' end, polyA nucleotide sequence at 3' end. Poly A addition site is 18-20 bp downstream in mRNA transcript.
Explain how transcription is initiated.

What would a mutation in the promoter sequence result in?
Transcription factors form a transcription complex with RNA polymerase = initiates transcription.

Common promotor sequences are GC, TATA and CCAAT boxes. TATA box is 25 bp before initiation site.

Mutation = reduced transcription of coding region.
What are enhancers?

What do they cause?
Regulatory element located greater distance from potential initiation site (upstream 5', downstream 3'). Proteins bind to these and promoter sequence to permit sliding down DNA to locate promoter.

Cause tissue-specific expression of genes, only active in certain cell types.
List 3 regulatory elements other than enhancers.
Chromatin openers: allow certain genes to access RNA polymerase. Response elements: DNA sequences that facilitate binding of other regulatory elements, e.g. cyclic AMP. Silencers: like enhancers, but decrease transcription.
What are cis and trans effects?
Cis: cis acting locus refers to a DNA locus close to coding region that controls transcription. Trans: regulation by a protein coded for by a gene distance from the one being transcribed.
What 2 strands is DNA composed of?

Which one is mRNA the same as?
Coding (sense) and template (antisense) strand.

mRNA is the same as the coding strand, but uses U instead of T.
How are introns removed from the primary RNA transcript?

How can mutations affect this? Give an example of a mutation occurring this way.
Via process of splicing....splicosomes (composed of primary transcript, proteins, and 4 small nuclear RNA).

Controlled by consensus sequence in the parent DNA - common to all eukaryotes.

Consensus sequences (GT and AG) = GU and AG in primary transcript. GU = 5' end, AG = 3' end. These define a splice junction. A lariat is formed allowing sequential exons to come together.

Mutations can remove splice junctions or create consensus sequence to make new ones. E.g. thalassemias.
What are snRNA?

Where are they held?

What can go wrong?
Small nuclear RNA - components of the spliceosome held in ribonucleoprotein particles (RNP) called SNURPS!

Systemic lupid erythematosus (SLE) have antibodies to some snurps.
How is a genomic library made?
DNA sequence digested by a bacterial restriction enzyme that produces characteristic fragments of duplex DNA. When whole genome is digested with one restriction enzyme = creates a genomic library
What does blotting involve?

What is the difference between Southern, Northern and Western?
Blotting = pressing gel eletrophoresis and sheet together to have bands transfer to nitrocellulose.

Southern - blotting of DNA fragments
Northern - blotting of RNA
Western - blotting of protein bands using antibodies to identify proteins
Outline DNA sequencing using the dideoxy method.
1. DNA is isolated to ss.
2. Labelled dideoxy nucleotides are added.
3. Short, complementary primer added to 3' end.
4. 4 incubations are set up - DNA polymerase continues to add DNA to unknown, using it as a template.
5. Elongation stops once a dideoxynucleotide is incorporated.
6. Repeated with each ddATP, ddCTP, etc.
7. Varying length fragments are created, seperated by electrophoresis, and each is indicated by emission of radioactive particles, allowing DNA to be read.
What does a restriction enzyme do?

What can be done with sticky ends?
1. RE works as a dimer that recognizes a palindromic sequence.

2. Will generate either blunt or sticky end fragments. BamHI = sticky, Hpa I = blunt.

Sticky ends can hybridize with DNA ligase to join and form recombinant DNA.
What is the significance of having an RE cleave at methylated bases?

Explain how RE's can generate a characteristic fingerprint.

How can RE's identify mutations?
Cleaving at methylated bases can indicate inactive genes as methylation can be associated with inactive genes.

Mutations can be identified by changes in the recognition site for an RE. This creates a new RE, and the fragments made by a RE digest will change.

A genome map can be cleaved at x # of sites by a given RE. This can be used to identify small genomic DNA or DNA fragments
What are plasmids?

What are BAC's?

What is a bacteriophage?
Plasmids are circular bacterial DNA, extrachromosomal, 1000 bp. Accommodates 10-15 kb.

BAC are artificial plasmids, 100-300 kb.

Bacterial virus, (lambda virus is key), contains linear DNA and acts as a vector, accomdating DNA inserts up to 20 kb.
What is a cosmid?

What is a YAK? What are the 4 key components? What are they used for?
Cosmid is hybrid between a plasmid and a bacteriophage that can accept larger inserts than either alone; 40-50 kb.

YAK - Yeast Artificial Chromosome. Constructed from: 1) a yeast centromere, 2) yeast sequences for the chromosome ends (telomere sequence), 3) selected markers and 4) cloned DNA in the megabase range (1000 kb). Used for large-scale cloning.
Outline a strategy for cloning human genomic DNA using the plasmid pBR322.

(Key: 5 Stages)
Stage 1: Cutting. pBR322 has resistnace genes for ampicillin and tetracycline. Plasmid is incubated with a restriction enzyme RE PstI which cuts it at a plasmid contained gene for ampicillin resistance (AMPr). Tetracycline resistance left intact. Human DNA is also incubated with PstI to make fragments with same ends.

Stage II: Incorporation: Mixing fragments with the plasmid will incorporate human fragments at complemtary sticky ends using DNA ligase.

Stage III: Transformation: E.coli infected with new plasmid (1/cell).

Stage IV: Cloning. Infected e.coli multiply and plasmids replicate. New cells are plated with agar and either ampicillin or tetracycline. Replicate plates are made y placing a filter on top of 1 plate with bacerial colonies and placing on fresh agar plate in same position. Each colony formed is a clone. Selection can be done for AMP sensitive, tetracycline resistant colonies to select the transformed cells. Incubation of the engineered plasmids with PstI will recover the DNA.

Stage V: Selection. Using a probe for the DNA of interest to find and identify the DNA of interest.

Drawback: need to have some idea of the fragment of interest to design probe to recognize foreign DNA.
What is a library?

What is a genomic library?

What can the fragments be incorporated into?
Library: a collection of recombinant clones based on DNA fragments. Used to make large quantities of specific DNA fragments via cloning.

Genomic Library: made from partial digestion of total DNA from cell or tissue with RE. Includes all DNA: exons, introns, etc.

Large fragments generated can be incorporated into bacteriophages, cosmids, BACs or YACs.
What is a cDNA library?

How is cDNA prepared from reverse transcriptase?

What are the advantages?
cDNA library: made from mRNA produced by a cell line or tissue. Reverse transcriptase generates the DNA exon sequence, generating DNA strand complementary to RNA strand which can be used to derive complimentary cDNA.

mRNA is used as a probe by RT. A short oligo primer is used and the DNA is synthesized by RT. RNA is removed by alkaline hydrolysis. ssDNA serves as its own primer and template for second strand synthesis by RT. This creates a hairpin loop that is removed using a single-strand-specific nuclease.

Advantages: cDNA library is smaller than a genomic library, easier to handle. Lacks non-coding introns. Represents all expressed genes in a cell type.
What is a probe?

What are the 3 types of probes?
Probe: single stranded sequence of DNA or RNA that is radioactively or chemically labeled. Probe sequence is complementary to portion of sequence found in cloned DNA. Do not have to know the complete aa sequence.

Genomic probe: amplified sequence of genomic DNA. Not expressed as gene. Double-stranded.

3 Types of Probes:

cDNA probe: amplified sequence, represents exon of expressed gene. Double or single strand. Generate ss cDNA to hybridize with genomic DNA fragment.

Oligonucleotide: probe made of short sequence of nucleotides. Single strand. Known aa sequence for protein of interest can generate this probe.
What is the difference between cDNA and genomic DNA sequencing?
Sequencing cDNA gives total exon sequence from which mRNA and protein is deduced.

Sequencing genomic DNA gives introns and regulatory sequences in gene.
What are the differences between genomic and cDNA libraries?
Genomic: constructed from genomic DNA, cDNA libraries start from mRNA expressed from a tissue.

Genomic = all sequences, cDNA = only exons

Genomic = all genes, cDNA = only expressed genes in tissue selected.

Genomic requires large vectors, cDNA requires small vectors (i.e. plasmids)

Genomic libraries are identical from all cells/tissues, cDNA differ between tissues depending on genes expressed.

Genomic constant throughout differentiation, cDNA may vary depending upon differentiation status.
What is probe degeneracy?

Explain how antibodies can be used as probes? When may this not work?

What is an expression vector?
Probe degeneracy: covers variation in probe sequence by generating more probes to cover aa's with more than one codon.

Antibodies are used to find the actual protein generated by transcription. May not work if the bacteria cannot perform translational modification on the produced protein.

Bacteria that produce new proteins as a result of insertion of a cDNA sequence
How does blotting work?
Involves separation of macromolecules by electrophoresis and their transfer, by blotting, to nitrocellulose sheets where the identity of separate parts can be checked using a probe.
What is a Southern blot?

What is it used for?
Used to identify a change in gene structure, as the cleavage pattern of a restriction enzyme. This could make a smaller DNA fragment.

Used for detection of a point mutation (i.e. loss of a restriction enzyme site), DNA deletion, resulting in a smaller DNA fragment. DNA translocation will also alter RE pattern if chromosomes are swapped between arms, gene amplification increases # of genes for a particular protein.
What is Northern blotting?

What is it used for?
Used to detect RNA. RNA is separated, blotted onto nitrocellulose, and hybridized with an RNA or DNA probe.

Use to detect absence of an mRNA species, i.e. if a gene is not expressed, then mRNA absent. Can also be used to detect deletions, which would result in smaller RNA. Used to detect increased levels of mRNA, i.e. in inflammatory response, or RNA size changes - if introns were not lost, size would be larger.
What are the differences between genomic and cDNA libraries?
Genomic: constructed from genomic DNA, cDNA libraries start from mRNA expressed from a tissue.

Genomic = all sequences, cDNA = only exons

Genomic = all genes, cDNA = only expressed genes in tissue selected.

Genomic requires large vectors, cDNA requires small vectors (i.e. plasmids)

Genomic libraries are identical from all cells/tissues, cDNA differ between tissues depending on genes expressed.

Genomic constant throughout differentiation, cDNA may vary depending upon differentiation status.
What is probe degeneracy?

Explain how antibodies can be used as probes? When may this not work?

What is an expression vector?
Probe degeneracy: covers variation in probe sequence by generating more probes to cover aa's with more than one codon.

Antibodies are used to find the actual protein generated by transcription. May not work if the bacteria cannot perform translational modification on the produced protein.

Bacteria that produce new proteins as a result of insertion of a cDNA sequence
How does blotting work?
Involves separation of macromolecules by electrophoresis and their transfer, by blotting, to nitrocellulose sheetes where the identity of separate parts can be checked using a probe.
What is a Southern blot?

What is it used for?
Used to identify a change in gene structure, as the cleavage pattern of a restriction enzyme. This could make a smaller DNA fragment.

Used for detection of a point mutation (i.e. loss of a restriction enzyme site), DNA deletion, resulting in a smaller DNA fragment. DNA translocation will also alter RE pattern if chromosomes are swapped between arms, gene amplification increases # of genes for a particular protein.
What is Northern blotting?

What is it used for?
Used to detect RNA. RNA is separated, blotted onto nitrocellulose, and hybridized with an RNA or DNA probe.

Use to detect absence of an mRNA species, i.e. if a gene is not expressed, then mRNA absent. Can also be used to detect deletions, which would result in smaller RNA. Used to detect increased levels of mRNA, i.e. in inflammatory response, or RNA size changes - if introns were not lost, size would be larger.
What is Western blotting?

What is dot blotting?
Identifies proteins on the basis of labeled antibodies. Allows identification and quantization of specific proteins. Also known as an immunoblot or immunoassay.

If the sequence of a mutated DNA sequence is known, an allele-specific oligonucleotide can be designed. A probe for the normal DNA sequence is also made. DNA is not separated by electrophoresis, and is instead just blotted. Total DNA is exposed to the probe, allowing for id of homozygous or heterozygous for mutant.
What is pulse field gel electrophoresis (PFGE)?
Normal gel electrophoresis = fragments up to ~100 000, PFGE allows for separation of larger fragments by periodically changing the orientation of the electric field, putting larger fragments on a snake-like path. This simplifies sequencing.
What is a PCR?

How is it performed?

What are limitations?

How does this differ from RT-PCR?
PCR = polymerase chain reaction - "molecular photocopying" - production of large amounts of DNA from small quantity of DNA without use of cells.

dsDNA is heat denatured to ss and primers are put onto opposite ends of target regions. Primers are extended using DNA polymerase. Heat denaturation occurs again, leading to another round of extension.....

Limitations: contamination.

RT-PCR: RNA is used to generate cDNA which is amplified by PCR. This is used to amplify the cDNA.
What is karyotyping?

What are the 2 types of karyotyping? Describe.
Analysis of the set of chromosomes.

In Situ Hybridization probes for a particular DNA sequence labeled with tritium when chromosomes are in metaphase. Gives an idea of the position of a gene on a chromsome. Drawback: low resolution.

Fluorescense in Situ Hybridization: fluorescent labeled probe used is instead. Better resolution, faster.
What are Restriction Fragment Length Polymorphisms (RFLP's)?

Use?
Populations have variances in DNA sequence; this may change site for a restriction enzyme = different fragment lengths in different individuals. After RE digest and southern blot, probe can show characteristic banding pattern for individuals.

If an RFLP is linked to a genetic disorder, it can be used for prenatal diagnosis or to id carriers of x-linked disorders. RFLP markers are usually found on the same chromosome near the dx gene = markers and gene are inherited together. If dx gene is unknown, id of RFLP can help id location of dx gene.
Give 3 causes of DNA polymorphisms.
1. nuclotide substitutions (mutations)
2. small deletions or insertions of DNA
3. presence of variable number of repeated DNA sequences
How are minisatellites and microsatellites used in gene-mapping?

What are single nuclotide polymorphisms used for?
Minisatellite - a tandem repeat of 20-120 bp of DNA. Not transcribed into RNA. Can have variation in the minisatellite repeats. Application: Variable Number of Tandem Repeats Polymorphisms VNTR.

Microsatellite: smaller repeat units of 2-5 bp in tandem. In the population, there is variation in the microsatellite repeats at differ points in the DNA = different RE digest. Individual DNA "fingerprint" depends on the nature of variation in repeats throughout the genome.

SNP's = genetic markers.
What is chromosome walking?

How is it performed?
A method of genome sequencing where cones carrying overlapping sequences of DNA are sequentially isolated.

Gene X is isolated from a large piece of DNA. Although the exact location is not known, a probe can find this. A library with a series of overlapping DNA fragments is used. THe probe will hybridize with clones containing fragment 1, which can be isolated, sequenced, and used as a probe to find fragment 2. Repeated until entire sequence is found.
What is chromosome jumping?
Permits cloning of DNA sequences 100-500 kb away from probe start site. A jumping library is made by taking a large fragment of DNA, circularizing these to bring the sequences of interest together, and making a conventional library with linear fragments with the now more closely placed DNA site. Used when walking from one end to the other may be difficult.
What are 3 strategies to hunt for a disease gene?
Functional approach, Candidate gene approach, Positional cloning.

Functional approach: gene is identified by the biochemical defect without referring to chromosome position.

Candidate gene approach: protein with a known function is used to detect differences when there is a loss of the functional gene producing the protein.

Positional cloning: functional gene product is not known, and isolated is based on finding the gene in the chromosome. This information is found from linkage analysis of multiple affected families. E.g. CF: studied markers flanking gene. Found on CR 7. E.g. Duchenne's muscular dystrophy: gene was found on X chromosome.
How can a gene be identified in a long tract of DNA?
1. Look for cross-hybridization of the DNA between species. Conservation could = gene.

2. Look for deletions or rearrangements in the DNA sequence as compared to controls.

3. Look for CpG islands on the 5' side of vertebral genes.

4. Check for absence of stop codons - genetic sequence would be free of codons normally signaling an end to translation.

5. Isolate an mRNA sequence matching the gene.
How was the CF gene identified?
Linkage analysis was used to find the mutant gene. The nature of the defective protein was not known.

Researchers assessed DNA from families where dx was found. Found MET marker on CR 7, then started walking and jumping.

Fragments isolated during walking and tested to determine whether these would bind to a cDNA library made from a CF sweat gland. Found hybridization between DNA and cDNA library member. A flanking RFLP was also found on the 3' side of the gene.

The CF gene was sequenced and the gene was located precisely on the chromosome.
What is the CF mutation?

What does the gene normally code for?

Pathology?
A deletion of 3 base pairs encoding for phenyalanine reside at position 508 in the protein. There are over 850 mutations leading to CF, but f508 is most common.

CF gene codes for a membrane protein CFTR (cystic fibrosis transmembrane conductance regulator). This spans the plasma membrane 12x and had binding domains for ATP.

Lost of phenylalanine changes shape of CF protein.

Pathology: the protein is functional, but is destroyed by quality control mechanisms. As such, it never gets to the plasma membrane.
What is the positional candidate approach?
A chromosome region may be indicated by linkage studies or the nature of known genes and their proteins. This region is then surveyed for candidate genes.

E.g. Marfan's syndrome - mapped to chromosome 15q was the protein fibrillin was mapped to this area (Marfan's = fibrillin defect).
What is gene therapy?

What are 3 approaches?

What is a key concern?

Example of success?
Attempt to cure dx by putting a functional gene into the genome to code for the protein. Done for somatic cells.

3 Approaches: 1. Gene replacement (mutant gene is replaced in a normal gene). 2. Gene correction: correction of mutant dx gene. 3. Gene augmentation: functional gene is introduce to genome to compensate.

Concern: insertional mutagenesis: insertion of new gene into another functional gene.

Example: SCID (severe combined immunodeficiency disease) caused by deficiency in enzyme adenosine deanimase.
What does gene knockout involve?
Determines the importance of a gene (hence protein) by removing function. Shows whether defective gene is responsible for a dx.

May not work in different species, other functional proteins may compensate.
What is RNA interference? Explain.
Eliminates the function of a specific gene. Worm C. Elgans is fed e.coli engineered to produce a specific dsRNA, the C. elgans takes up the RNA.

The dsRNA is broken down by an enzyme called dicer into smaller dsRNA. This is then split into ssRNA, one of which is small interfering RNA (siRNA).

siRNA is complexed with an enzyme that guides it to complementary mRNA which is hydrolyzed and neutralized.
Explain how transgenic animals are used in recombinant DNA techniques.
Foreign DNA is incorporated into the genome of animals to transmit altered DNA to offspring. These genes are injected into fertilized mice ova to make transgenic animals to pass new genes to offspring.
What is site-directed mutagenesis?
A specific mutation is introduced and the altered DNA sequence is introduced to bacterial cells using vectors, leading to altered protein production by the bacterial host. THis allows evaluation of the importance of a single aa in the protein structure.
Describe the human genome project.

Which was the first chromosome completed? What was found?

What was found on chromosome 21?
Project to completely sequence the human genome with known and unknown genes, pseudogenes, markers and tandem repeat areas, mapping all 24 chromosomes.

Genome contains 3 x 10^9 bp, 32 000 genes of which 1-1.4% of genomic DNA is translated to protein.

Chromosome 22 was first completed. FOund imunoglobin-lambda gene cluster, Bcr gene involved in some forms of leukemia, mutations responsible for Ewing's sarcoma and giant-cell fibroblastoma.

21: AAP protein (amyloid precursor involved in Alzheimer's dx), SOD 1 gene for superoxide dismutase. Involved in Down's syndrome in trisonomy 21.
What is involved in mapping a chromosome?
Use of a genetic map to give hypothetical markers and the distances between them, the cytogenic map which gives the banding pattern for a chromosome, the physical map which gives the physical locations of gene markers along the sequence, the restriction map which shows the points of RE action, contig map (overlapping DNA sequences) and the sequence tagged sites (STS) map - which is small areas of known sequence.
How does diagnosis via gene chips or oligonucleotide array detectors work?

What is a complication?
The chips have oligonucleotide probes in seperate discrete squares on each chip face. Can be used to identifiy gene expression in cells: the mRNA population for the proteins synthesized in the cell are isolated and a cDNA library is constructed. THese are tagged with dye and the sampe is run over hte chip. The probe can be designed to match a specific marker sequence with known DNA coding for proteins. the cDNA will bind with a specific square in the chip.

E.g. could be used to determine response to ischemic stress.

E.g. 2: cDNA could be constructed from population of cells from a child suspected of a disorder, tagged with probes for specific, known mutations on genes.

Complication: possible duplication of sequences within DNA = better to use cDNA libraries instead of genomic libraries.
What is the 2-hit model?

Outline 2 ways the BRCA1 protein can be mutated.
A carrier for a defective gene can sustain a mutation in the matching functional gene (Aa --> aa). When one gene is dysfunctional and is passed on by inheritance, it is easier to sustain damage to the second functional allele.

BRCA1 protein is large, and over 150 mutations have been found. Some are 1) point mutations and 2) some are frameshift mutations, where the deletion or insertion is eliminated in the triplet coding for aa downfield from the frameshift. This truncates the protein product of the mutated BRAC1.
How does BRCA1 work?

How does BRCA2 work?
BRCA1 is involved in the activity of the enxyme (cyclin-CDK) involved in the control of the cell cycle. If BRCA1 is damaged, the CKD is switched off and the CKD arrests the cell cycle, shutting down mitosis until DNA damage is repaired.

BRCA2 risks are associated with cancer in both sexes, and breast, ovarian, pancreatic, hepatic and prostate cancers. Also involved in gene repair and tumor suppression. Promotes homologous recombination - DNA repaired by the removal of damaged DNA; impaired function