Western Blot Analysis

Proteins isolated from cell extracts are separated according to size and charge by polyacrylamide gel elecrophoresis, transferred to a membrane and detected by antibodies.

What methods do we use to detect copy number changes (deletions and duplications in genomic DNA)...

Just give the 4 names of the tests.

1. Quantitative PCR
2. Multiplex ligation-dependent probe amplification (MLPA)
3. Fluorescence in situ hybridization (FISH)
   
4. Array comparative genomic hybridization [a(CGH)]

What are 3 kinds of expression (RNA) testing?

What research and clinical uses do they have?

1.  Northern Blot

2.  Reverse Transcriptase PCR (RT-PCR)

3.  Expression Arrays

Research:

1.  Characterize diseased and normal tissue to elucidate molecular pathogenesis.

2.  Identify genes associated w/ a disorder, assuming that genes w/ altered expression relative to control tissue play a role in that disease.

Clinical:  Characterize tumors for prognosis and match the specific chemo w/ the specific type of tumor.

a(CGH)

1.  What it stands for

2.  What it's used for

3.  How it's done

1.  Array comparative genomic hybridization

2.  Method to detect copy # changes in genomic (g) DNA.  Can detect deletions/duplications that are ~100 kb or larger in size, looks at entire genome.

3.  Patient and control DNA's labled with different fluorescent molecules, then co-hybridized in equal amts to the array of DNA probes.  Slide is then imaged by computer.  Normal karyotype - each probe hybridizes equally to test *green* and reference *red* DNA.  Result is yellow, 1:1 ratio.  Deletions/duplications - change in the ration between red and green.

FISH

1.  What it stands for

2.  What it's used for

3.  How it's done

1.  Fluorescense in situ Hybridization

2.  Method to detect copy # changes in gDNA.  Detects deletions that are for ~100 kb to several Mb in size, but only a few loci are tested at a time.

3.  Performed on metaphase chromasomes. Chromasome DNA is denatured on a microscope slide, then hybridized with a fluorescent probe.

MLPA

1.  What it stands for

2.  What it's used for

3.  How it's done.

1.  Multiple ligation-dependent probe amplification

2.  Method to detect DNA copy # changes in gDNA

3.  a)  1/2 probes hybridize next to each other at their complementary target regions in gDNA.

    b)  Probes joined by ligase, amplified by PCR using common adapters.

    c)  Amount of final PCR product for each probe corresponds to amount (copy #) of the target region in the gDNA.

Quantitative PCR

1.  What it's used for

2.  How it's done

1.  Used to detect copy # changes

2. a)  DNA or RNA of interest is amplified w/ fluorescent probe or DNA-binding dye.  Intensity of fluorescent signal generated increases in proportion to the amout of PCR product produced.

    b) Fluorescent signal intensity measured is then graphed on an amplification chart to determine CT (point when fluorescence signal, or PCR product, is first detected).

     c)  CT values are compared against a standard curve to determine the amount of DNA or RNA in a patient's sample.

Example of a PCR stand alone test:

- The forward primer is designed at it's 3' end to match allele B, but not A.  If B is present in target DNA, an amplicon will be produced in PCR replication. A complimentary ARMSPCR is also performed; the forward primer is designed to match A allele and not B.

PCR

1.  How it's done

      2.  What it's used for

1.  Produces millions of copies of short DNA through repeated cycles of:

denaturation

annealing

elongation

2.  a) generate enough DNA to perform a test like sequence analysis or mutation scanning; b) be a test unto itself, like allele specific amplification or trinucleotide quantification; c) good for diseases with common mutations

Kinds of nucleotide substitutions:

Missense mutations:  aa substitutions

Nonsense mutations:  premature stop codons

RNA processing mutations

Splice site mutations

regulatory mutations

As mRNA leaves Pol II, the cap is added to the 5' end by factors bound to Rpb-CTD.

Cap is made of methylated guanosine residue, attached backward to the first mRNA nucleotide, 5' to 5' triphosphate linkage.  This protects the mRNA from degredation. 

Cap is also a binding site for proteins that facilitate:

Splicing of pre-mRNA

Transport of the mRNA to the cytoplasm

Translation of the mRNA by ribosomes

Where ribosomal DNA is transcribed into rRNA.

Large, dark staining region near the center of the nucleus.

Consists of up to 10 chromasome tips, each of which have a nucleorlar organizer of ~ 40 rRNA genes each, giving up to ~400 rRNA genes/cell.

Organizers start as several small nucleoli, coalese into a single nucleolus. 

Prominent during interphase.

Meiotic error in which homologs (meiosis I) or chromatids (meiosis II) fail to separate, resulting in abnormal chromasome segregation on the gametes.

A.  Full Monosomies:  virtually always lethal.  Monosomy X is a partial exception.

B.  Full Trisomies:  Generally lethal.  Exceptions:  13, 18, 21, sex chromasomes.

• Multiple congenital abnormalities
• Presumed Downs, other well known autosomal trisomy
• Genital ambiguity
• Primary amenorrhea
• Short stature in females
• Small testes, delayed pubery in males
• Stillborn infant
• Recurrent stillborns
• Prenatal diagnosis assessment
• Indentification of translocation carriers

Klinefelter Syndrome

Diagnosis

• Incidentally on amnio/CVS
• Phenotype at puberty
• Infertility in adulthood
• Some go undiagnosed

Klinefelter Syndrome

Genetics

• Incidence:  1/600 - 1/1000
• Mosaic (eg:  46, XY / 46, XYY) - 10%
• Poly-X (eg:  XXXY) ~10%
• Meiotic nondisjunction, maternal > paternal (slight)
• Weak maternal age effect (vs. Down)

Kleinfelter Syndrome

Growth and Development

• Normal through age 5, then tall with long legs
• Slight tendency for delays in motor/language development, mild reading difficulties - MOST DEVELOP NORMALLY.
• Sensory-motor integration problems for school age
• Verbal skills lag non-verbal skills by age 4
• Slightly greater than 50% mild reading difficulty
• Avg verbal IQ:  90
• Avg IQ:  103

Kleinfelter Syndrome

Medical Issues

• Malignancy:  Breast cancer more common than in 46, XY males, mediastinal germ cell tumors < 1%.
• Cardiovascular:  Increased risk of mitral valve prolapse, DVT, leg ulcers, hypercholesterolemia
• Immunologic:  Increased lupus, diabetes, rheumatoid arthritis, thyroid disease.

Klinefelter's Syndrome

Dispelling Myths

NO

Consistent alteration of body fat distribution

Increase in aberrant sexual behavior

Increase in homosexuality

Turner Syndrome

Birth Defects

• Congenital heart disease
• Kidney structural abnormality
• Lymphedema
• Cystic Hygroma (jugulolymphatic obstruction sequence)
• Nuchal redundancy

Turner Syndrome

Natural History

• Small stature not detected until after 5, secondary to SHOZ hemizygosity
• Ovarian failure leads to failure to pubertal development, primary amenorrhea, infertility
• Development - NOT associated with retardation but mild learning disabilities, including impairment of visual/spacial functioning.

Turner Syndrome

General Information

• Synthesis of DNA and RNA
• Energy metabolism (ATP)
• Intracellular signaling (cAMP, cGMP, GTP)
• Coenzymes (especially adneosine derivitives)
• Activating metabolic intermediates (eg. aminoacly-AMP)

It's usually hydrolyzed to hypoxanthine, ammonia and ribose-1-phospate by the following reaction in RBC's:

         H20 ->Pi               H20 -> NH3

AMP -------------> A---------->

        phosphatase           adenosine deaminase  

    Pi -> ribose 1-P

I ----------------> hypoxanthine

   nucleoside phoshorylase

1.  DNA pol stops at site of damage, DNA synthesis arrested, leading to apoptosis.  DNA POL ARREST

2.  DNA pol falls off site of damage, special DNA pol for that kind of damage comes on and inserts the right nucleotide opposite the site of damage.  Replicative DNA pol hops back on - no mutation.  (ERROR-FREE BYPASS)

3.  Special DNA pol inserts random nucleotide opposite of site of damage, mutation created.  Most dangerous. (ERROR-PRONE BYPASS).

Important mechanisms of DNA damage

Depurination

Spontaneous hydrolysis of glycosidic linkage between ribose and A or G.  Induced by alkylating agents like chemo drugs.  Loss of G most common.  Special DNA Rev 1 inserts dCMP opposite missing base, usually averting a mutation.

Important mechanisms of DNA damage

Deamination

Mostly cytosine.  Occurs spontaneously @ low frequency, increased in rate by some chemicals.  Deaminated bases have altered pairing properties, can cause substitutions upon replication.  EX:  Deamination of C creates U, which pairs with A rather than G.  Repair pathway exists to remove U from DNA.

Important mechanisms of DNA damage

Ultraviolet (UV) Radiation

Important mechanisms of DNA damage

Oxidation

Important mechanisms of DNA damage

Ionizing radiation

High-energy radiation generates double-stranded breaks in DNA.  Resulting free ends highly reactive, participate in rearrangements, including inversions and translocations.  No DNA pol can replicate past double stranded breaks, but they can be repaired by homologous recombination or non-homologous end-joining (NHJE) proteins.

1.  Portion of hte damaged strand is removed, exposing a single-stranded region that can be as short as one nucleotide or long as hundreds.

2.  DNA pol fills in damaged strand adding dNMPs to free 3' OH of gapped strand

3.  DNA ligase seals the nick left between the 3' end of newly made DNA and 5' end of the gap, using ATP.

An enzyme - uracil-DNA glycoylase - recognizes & hydrolyses the U from deoxyribose.  AP endonuclease then recognizes the abasic site, initiates its repair as in depurination.  Use of thymine rather than uracil in DNA allows the detection by the DNA repair enzymes.

Non-hereditary non-polyposis colorectal cancer.  One gene responsible codes for protein called MSH2, that mediates repair of mismatched bases.  Autosomal dominant, but can't get cancer unless both alleles is affected. 

Mutations in genes involved in mismatch repair (MSH6, MLH1, PMS2) account for most other cases.  Cancer likely results from mismatch repair pathway.