Molecular Genetics Quiz 3

Southern Blotting uses restriction enzymes. Northern Blotting doesn't use restriction enzymes because enzymes don't cut RNA.

1. immobilized targets are proteins
2. (utilizes the theory similar to Southern blots)
3. According to the molecular weights of various proteins, target proteins are SEPARATED on SDS-polyacrylamide gels (SDS-PAGE)
4. SDS is a denaturing reagent that makes all proteins uniformly negatively charged such that the rates of migration are solely depends on their sizes
5. Proteins in the polyacrylamide gels are next blotted to a nitrocellulose membrane and then fixed
6. Protein-containing membrane is future probed with protein-specific antibody, in order to detect the protein of interest
7. Detection of antibodies can be carried out by ELISA

is a sandwich reaction where you'll use a primary antibody to bind to a protein of interest then you use the secondary antibody will catch on the primary antibody where the substrate would bind to the secondary antibody - there is chemiluminescent.

Western Blots - use a polyacryamline gel

Northern & Southern - use agarose gel

You can do a Dot Blots or Slot Blots. (quicker than Southern Blotting)

1. GOOD THING: you can analyze simultaneously on a dot or slot blot.
2. Can test for only ONE GENE or gene product or address one kind of genetic aberration
3. MULTIPLE SPECIMENS can be analyzed for ONE GENE.
4. technologies are applied to analyze expression, mutation, amplification and deletion

• Extracted DNA is denatured and the ssDNA is bound to a positively charged nylon membrane
• After the DNA is bound to the membrane, a probe complementary to the gene of interest is applied and allowed to hybridize to the DNA
• the hybridized complex is detected by Colorimetry or chemiluminescence methods
• The amount of DNA in the sample is estimated by comparison of the density of the band or bands observed to that of the standards

• a dot blot method utilizes the theory similar to Southern blots. Specimen DNA was first amplified by PCR, immodilized on a pair of membranes, and then each membrane is respectively.
• Utilizes the differential outcomes in hybridization between target DNA and the probe with short sequences of about 20 bases. Failed hybridization is due to one or two mismatches, and it can be distinguished from those with excellent hybridization due to a "perfect match".
• Probes used in this assay are synthetic single-stranded with normal or mutant target DNA sequences.
• At specific annealing temperatures and conditions (stringencies), given probes wil NOT bind to one or two mismatached based. Therefore, probes display differential binding to either mutant or to wild-type target DNA sequences.
• Applications include: detection of BRCA1 and BRCA2 genees in inherited breast cancer.

You can amplify a gene. You can copy the DNA - only make 2 DNA strands at the end. The primer will find the corresponding DNA.

dot blots is when you compare the wild-type probe to the mutant probe.

FOR example,

Wild-type = GAG is glutamate

Mutant = GTG is valine (sickle cell anemia)

• varieties of mutant or normal probes are respectively immobilized on each WELL of a 96-well plate
• specimes are in the LIQUID PHASE
• Multiple genes within a specimen can be simultaneously analyzed, based upon the respective probe immbolized on each well.
• After hybridization between the probes and target specimens, if perfect match duplex can be formed between the probe and target specimens, signals can be generated upon the addition of substrates.
• Applications include: infectious disease testings, detection of mutation in factor V Leiden, and HLA tissue typing
   

• Use to test Cystic Fibrosis and Lymes disease
• For genotyping pathogenic viruses: HPV, HIV, HCV, and HBV
• It is a multiparameter assay for simultaneous detection of different allelic forms of the same gene (locus)
• Reagents needed:
  • Unlabeled probe with known sequences are immobilizaed onto membrane strips.
  • Specimen DNA (or RNA) isolated from patients are amplified by PCR such that the biotinylated deoxynucleotides could be incorporated into the final amplified products
  • The binding (hybridization) between the biotinylated PCR products and the matched probes could generate signals for detection.

1. Denature the ds biotinylated PCR product
2. Hybridize it with specific oligonucleotide probes that have been immobilized as parallel lines on membrane-based strips
3. Add streptavidin that would capture the biotinylated hybrids.
4. Add alkaline phosphatase.
5. Incubate with substrate (called NBT/BCIP) to generate purple/brown precipitate
   1. NBT - nitro blue tetrazolium chloride
   2. BCIP - 5-Bromo-4-chloro-3-indolyl phosphate, toluidine salt
6. Stop reaction with wash steps and record patterns

1. Macroarray utilizes theory similar to reverse dot blots: many different probes are immobilized on the membrane like nitrocellulose membrane
2. Specimen is "labeled" and is subjected to hybridization with many immobilized probes
3. Each array checks one sample at a time to appraise the possibility of aberrations occurring at multiple points of interest (dictated by the probes)
4. This assay requires a large quantity of nucleic acids in the specimen which can be sometimes achieved by PCR or by other amplification methods

1. Instead of nitrocellulose or nylon used in MACROARRAY, Microassay uses treated glass (called chips) on which very small targets (probes) can be immobilized.
2. Automated depositing system can place > 80,000 spots on a glass substrate.
3. Probes (unlabeled) are spotted on slides:
   1. cDNA
   2. PCR products
   3. RNA
4. Specimen (labeled, floating in the liquid phase)
   1. cDNA
   2. Genomic DNA
   3. RNA

1. In practice, the terms of macroarray and microarray are often interchangeable.
2. These two arrays may be differentiated according to spot sizes or the number of spots on the solid support
3. Macroarray: hundreds to thousands of probes on spots immobilized on nitrocellulose or nylon membranes
4. Mircoarray: tens of thousands of probes on spots coated on treated glass slides
5. Many other terms exist for naming gene arrays, including biochip, DNA chip, GeneChip (a registered trademark of Affymetrix, Inc., and a DNA array.

Microarray Probes

1. cDNAs (from libraries or PCR products0
2. Oligonucleotides
3. Genomic DNA fragments

Applications

1. Gene expression studies
2. Oligo-mediated sequencing
3. Genetic mapping studies
4. Mutation analysis
5. Polymorphism analysis

1. Screen the genome or specific genomic loci for deletions and amplifications
2. Genomic DNA is isolated, fragmented, may or may not be amplified and labeled for hybridization on the microarray slides (also called chips)
3. Prove higher resolution and more definitive genetic information

red = normal expression pattern

green = tumor expression pattern

yellow = commonly expressed genes

there are two different analysis: independent hybridization or comparative

1. Technology using sequence-specific nucleotide termination
   1. Maxam & Gilbert DNA Sequencing
   2. Chain termination
2. Strategies for detecting signals: Fluorescent vs radioactive
   1. labeled primers
   2. labeled dideoxynucleotides
3. Platform
   1. Manual
   2. Automated

1. There are four chemical cleavage reactions at the core of the Maxam and Gilbert sequencing system
2. The DNA to be sequenced must first be labeled, and then divided into four tubes each with respective reaction
   1. G: Add 200 µL of 50 mM sodium cacodylate, pH 8, 1 mM EDTA. Mix well and add 1 µL Dimethyl Sulfate (DMS - toxic) and incubate at 25°C for 4-5 minutes.
   2. G+A: Add 25 µL of formic acid, mix well and incubate at 25°C for 4-5 minutes.
   3. C+T: Add 10 µL H₂O and mix well. Add 30 µL Hydrazine and incubate 25°C for 7-9 minutes.
   4. C: Add 10 µL of 2.5 M NaCl and mix well. Add 30 µL of Hydrazine (toxic) and incubate at 25°C for 7-9 minutes.
3. Cleavage of the above products by using piperidine.

Deoxy - has a 3' OH that would help extend the sequence

Dideoxy - has a 3' H that would stop the reaction from building the sequence

- the single-stranded DNA molecule that is being sequenced

- there was a oligonucleotide primer for DNA polymerase that help starts the sequencing

- there were a lot of normal deoxyribonucleoside triphosphate precursors (dATP, dCTP, dTTP, & dGTP)

- there is a small amount of one dideoxyribonucleoside triphosphate (ddATP, ddCTP, ddTTP, & ddGTP)

- rar incorporation of dideoxyribonucleotide by DNA polymerase blocks further growth of the DNA molecule

1. incorporation of normal nucleotides (all four A, T, G, C) as well as 2,3-dideoxynucleotides (one in each reaction) by DNA polymerase, at an un-biased fashion
2. incorporation of 2,3-dideoxynucleotides into the growing DNA chain results in a termination of elongation reaction

A. double-stranded DNA is separated

B. the ss-original sequence run from 3' to 5'

C. the primer running from 5' to 3'

D. excess dATP, dCTP, dGTP, dTTP are added

E. in four separate vials - ddATP, dCTP, dGTP, dTTP with DNA polymerase separately to stop the reaction

F. then it was put in a gel electrophoresis

1. Ulitize the principle of "sequencing" by DNA synthesis
2. First step: denaturing double-stranded DNA into two of single-stranded DNA
3. one of the single-stranded DNA serves as a template for the subsequent sequencing reaction.
4. a primer is annealed to the complementary sequence of single-stranded DNA template to be sequenced.
5. DNA polymerase extends the primer using nucleotide mix (dNTPs) plus one of four ddNTPs (lack a 3' hydroxyl group thus diable the formation of a phosphodiester bond next to the dNTP and cause chain termination)

- a total of four reactions are required, each with one kind of ddNTP, meanwhile keep the remaining ingredients the same.

- products of different lengths are size-fractionated by electrophoresis

- short fragments that migrate fast and to the lower part of gel correspond to the 5'-end sequence while the large fragments that run slow and to the upper part of gel refer to the 3' end sequence. Thus, the sequence shall be read bottom-up (5' to 3')

- DNA sequence can be evolved by the (a) length of the products and (b) which of the four ddNTPs added to the individual reaction

- Detect the reaction end-products by the signals released from either the labeled primers or from labeled ddNTP (each ddNTP is labeled with its unique color). While the former is for manual operation, the latter is widely used for automation.

reaction comprises "all four" of the dideoxynucleotides (A, G, C, T) each with a different fluorescent color. Read the colors from bottom to top: TGCGTCCA-etc.

1. High throughout automatic system utilizes dideoxynucleotide sequencing method via electrophoresis

2. Detection of fluorescently labeled products at a real-time manner

Detection and data collection

1. Fragments are separated by sizes (not by shapes) and then captured by time elapsed
2. Denature condition in the capillary gel electrophoresis can be achieved by adding formamide and heat
3. Smaller fragments migrate faster and thus come off sooner
4. Four kinds of ddNTP and each is labeled with a different fluorescent color
5. Fluorescent fragments are detected by laser sensor
6. Raw data is converted into colored peaks

**Pyrophosphate is generated after each addition of deoxyribonucleotide to the growing strand DNA

- starting nucleic acids are single-stranded DNA templates

- unlabled dNTPs are added ONE AT A TIME

- DNA polymerase incorporates one of four dNTPs if complementary to the next base

1. If 1st dNTP is complementary tot he sequence of template, dNTP is incorporated to the growing strand and thus pyrophosphate is generated.

2. ATP sulfurylase uses pyrophosphate and adenosine 5' phosphosulfate to create ATP

3. ATP is used by luciferase to convert luciferin to oxyluciferin which gives off light. The intensity of signal is proportional to the number of nucleotides that are compementary to the given dNTP

4. Apyrase degrades unused dNTP and ATP.

1. production of signals would indicate which nucleotide (out of A, T, G, and C) is the correct base
2. if the sequence contains repeated nucleotides, the intensity of the signals (the height of the peak) would be multiplied according to the number of repeats.
3. the 2nd dNTPs are added and process repeats itself. The process is repeated with each of the four nucleotide added one-at-a-time to the reaction.
4. Real-time sequencing. This method doesn't need (1) labeled primers, (2) labeled nucleotides, or (3) gel electrophoresis

1. DNA polymerase - incorporates alpha phosphate in the dNTP to the growing strand of DNA and then releases a pyrophosphate
2. Sulfurylase - with the presence of pyrophosphate, it converts ADP to ATP
3. Luciferase - with the presence of ATP, it converts luciferin into oxyluciferin which then generates lights
   1. visible light is proportionate to the magnitude of ATP production and is indicative of number of repeated nucleotides in the sequence
4. Apyrase - degrades unincorporated dNTP and ATP