Download MCB Lecture 1 – Molecular Diagnostics

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What is Nucleic Acid Hybrdization?
o Denaturing DNA into two strands so that it can anneal with a new
strand.
What is a Southern Blot and how does it discriminate the sample?
o SNOW DROP
o DNA-DNA Hybrid
o Discriminates by size and sequence
What is a Northern Blot and how does it discriminate the sample?
o SNOW DROP
o DNA-RNA Hybrid
o Discrimination by transcript size and tissue distribution
What is in situ hybridization?
o RNA-RNA
o Localization of expression of a protein
What is a Western Blot?
o SNOW DROP
o Detection of Protein with Antibody after Electrophoresis
Case 12: Hemophilia is discussed below. Please answer the following
questions:
o What two genes are mutated?
 Factor VII
 Factor IX
o What is the Mode of Inheritance?
 X-Linked
o What are the three levels of severity?
 Severe
 Moderate
 Mild
o What type of mutation causes the most severe type?
 Null Mutation (Nonsense)
o A milder disease means that the F8 protein still has some residual
function. A more severe type means that the F8 protein has no
function. What proportion of cases is severe, moderate, and mild?
 Severe: 50%
 Moderate: 10%
 Mild: 40%
o In what specific way goes the F8 gene rearrange to cause Severe
Hemophilia?
 Intrachromasomal rearrangement
What are the steps of a Southern Blot?
o 1. Digest with restriction enzyme.
o 2. Gel electrophoresis
o 3. Fragments sorted by size on the gel.
o 4. Denature the DNA
o 5. Transfer to a plastic film
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o 6. Reveal the fragment of interest by hybridizing to a labeled probe
What are Restriction Fragment Length Polymorphisms (RFLPs)?
o A technique that exploits variations in homologous DNA sequences by
looking at the differences in locations of restriction sites.
o FAMILY SPECIFIC
Case 11: Sickle Cell is discussed below. Please answer the following
questions:
o What are some common symptoms of Sickle Cell Disease?
 Swelling of fingers
 Splenomegaly
 Low Hemoglobin
 High Reticulocytes
 Hemolytic Anemia
 Sickle-shaped RBCs
o What is a large reason for the distribution of Sickle Cell Disease in
Africa?
 Because having Sickle Cell disease gives protection against
Malaria
o What is the normal protein for Sickle Cell?
 HBA
o What is the Sickle Cell protein?
 HBS
o What is the gene called?
 HBB
o What specific protein is mutated?
 B-Globin (which forms hemoglobin)
o Can heterozygotes still show symptoms?
 Yes. Especially under stress.
o Why does sickle-cell anemia affect multiple different cell types
throughout the body (pleiotropy)?
 Because you have affects from the shape of the cells
themselves (ischemia, thrombosis, infarction) and you have
affects from the destruction of the cells by the spleen (anemia)
o What is the exact mutation that happens in Sickle Cell?
 Point Mutation
 AT
 GluVal
o What is unique about the area in which the point mutation takes
place?
 It takes place in a restriction site
What is the purpose of the Polymerase Chain Reaction (PCR)?
o In vitro amplification of specific DNA sequences
Does PCR require sequence information?
o Yes – just enough to make the primers
What do you need to perform PCR (ingrediants)?
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o Template DNA
o Primers
o dNTPs
o Buffer, Mg2+
o Thermostable DNA Polymerase (Taq)
What is the typical size of fragments that PCR can amplify?
o >1kb
How many cycles must you perform via PCR before you get the first exact
sample that you want to amplify?
o 4 Cycles
If you have a single base difference in sequence that does not affect a
restriction site, how do you detect it?
o Use PCR and then ASO probes.
o The mutation must be known, and it is suitable for screening
frequency mutations
PCR can only indicate the presence or absence of a target sequence, but no
the copy number. What is used to identify quantitative information about the
deletion or duplication?
o MLPA
What is MPLA?
o Uses primers designed to anneal head-to-tail on one strand of the
target exon.
o Stuffer (primer) is used
o Only if the primers anneal appropriately can DNA Ligase join them to
create a template for the standard primers to amplify
Case 9 – Turner Syndrome. Please answer the following questions about
Turner Syndrome.
o What is the Karyotype?
 Karyotype: 45X
 However, there can be a small proportion of 46XY cells in the
gonads
o What do you PCR?
 Y-specific sequence
What do you use to detect deletions under <10kb?
o Southern Blot
If you have a larger deletion, what do you use to detect it?
o Fluorescent in situ hybridization (FISH)
What type of probe is often used in FISH?
o BAC
What is Spectral Karyotyping?
o Chromosome painting
o FISH probes along entire length of the chromosomes so you can visual
rearrangements
Case 7 – 22q11 Deletion Syndrome. Answer the following questions:
o What do you use to detect 22q11 syndrome?
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 FISH
o What are some other names for 22q11 Deletion Syndrome?
 DiGeorge Syndrome
 Velo-Cardio-Facial Syndrome
o What are some symptoms?
 Short
 Cleft Palate, Prominent Nose
Case 13
o What are the symptoms?
 Nomal Karyotype
 Severe Mental/Growth Retardation
 Microcephaly
 Low-set ears
o What can you use to detect the mutation?
 Comparative Genomic Hybridization (CGH) – detects changes
in copy number only (deletions and duplications)
o If the CGH array of dots is below the normal line, what does this
mean?
 Deletion
o If the CGH array of dots is above the normal line, what does this
mean?
 Duplication
o Is case 13 a deletion or duplication?
 Duplication
o What can you use to replace BAC when doing a CGH, to increase
resolution?
 SNP Array
o Case 14 – Thalassaemia
 Symptoms?
 Hyperchroma in blood smear, many nucleated RBCs
 Marrow expansion in hands and skull
 Iron Overload in Liver
 What makes this different from Sickle Cell?
 Sickle Cell: Point Mutation
 B-Thalassemia: Null Mutation
What is DNA Sequencing?
o Gold Standard for mutation identification
o Can detect many Autosomal Dominant Conditions (newer)
DNA Sequencing uses Dideoxynucleotides. What are these?
o Lacks the 3’ OH group, which prevents extension of the chain once it is
added