Molecular Assays in Dx Testing

PCR

-Foundation for many molecular tests

-Amplifies targeted DNA fragment(s) using complementary oligonucleotide primers

-Cyles of denaturation, primer annealing, and primer extension replicate DNA frags

PCR product

Amplicon

PCR fragment size

Usually up to 1,000 bp

-if longer, the extension does not work properly

PCR primers target..

..unique DNA sequences in a sample

PCR primers hybridize..

..with the sample DNA and define the region of the DNA that will be amplified in PCR

Single site analysis

Amplify a single, unique target in the sample

Multiplexing

Amplifying several unique DNA fragments simultaneously within the same rxn

Allele-specific primers

1. Selectively amplify known alleles to distinguish mutant vs. wildtype alleles

2. Produce amplicons of different lengths for different alleles in sample

3. Presence/absence of amplicon can indicate

Electrophoresis- Definition

Separates DNA frags by size

-PCR products

-DNA fragmented by restriction enzymes

-Short tandem repeat (STR) typing

Electrophoresis- Technology

Permeable membrane

-Agarose gel or capillary tube

DNA is negatively charged and will migrate through the membrane toward the anode

Restriction enzymes form double-strand DNA breaks where?

At specific restriction sites

-Derived from bacteria

-Recognize and cleave palindromic DNA sequences

-Restriction sites located throughout genome

DNA polymorphisms located in restriction sites can alter cleavage patterns

Restriction fragment length polymorphisms

Real-time PCR- Definition

AKA melting curve

-Fluorescently labeled primers anneal to target DNA

-Fluorescence peaks only when primers bound to DNA

-Signal quenched when strands denature

Real-time PCR- Identify mutations

1. Annealing temperature shift due to change in nucleotide composition

2. Primers specific to WT or mutation

Real-time PCR- Primers

Primers are designed based on temperature

Real-time PCR- Quantitation

Quantitate the amount of DNA in the sample by monitoring fluorescence signal through PCR cycles

Triplet Repeat PCR

1. Forward primer hybridizes upstream of repeat

2. Reverse primer binds at 3' junction of repeat and randomly hybridizes throughout repeat

Varying frag lengths create what pattern on electrophoresis?

Stutter pattern

Fragile X - Normal alleles

< 45

Fragile X - Intermediate or gray zone

45-54

Fragile X - Premutation

55-200

Carrier

Increased risk for other FMR1-related disorders

Fragile X - Full mutation

>200

-Dx of FXS in male

-Increased risk of FXS in female

Southern Blot

-Identifies target DNA using a complimentary labeled oligonucelotide

-Does not require DNA amplification (PCR)

Southern Blot- Steps

1. Fragment all DNA

2. Electrophoresis on agarose gel

3. Transfer to membrane

4. Hybridize w/ labeled oligos

5. Expose to x-ray

Northern Blot

Targets mRNA

-Historically measures level of gene expression

-Less commonly used with the development of gene expression microarrays and RNA-seq

Western Blot

Targets protein

-Labeled antibodies identify proteins in a sample

Southern Blot for CGG Expansion in Fragile X Syndrome

1. Digest DNA w/ methylation specific enzyme (will cleave only unmethylated DNA)

2. Hybridize w/ FMR1-specific probe

3. Unmethylated CGG (digestion by restriction enzyme leads to short frag size)

4. Methylated CGG

-Resistance to digestion leads to long frag size (not cleaved)

Methylation Specific PCR- Process

DNA methylation at the "C" at 5'-CpG-3' dinucleotides inhibits gene expression

1. Gene regulation during development

2. Imprinting

3. X chromosome inactivation

4. Tumor suppressor gene silencing

Assays methulation status of CpG sites in CpG islands

Treat with bisulfite

Unmethylated C

C --> U

Methylated C

No change

Methylation Specific PCR- Primers

Target both methylated and unmethylated sequence

Methylation-Specific PCR in Angelman

Syndrome

1. Assay SNRPN gene in PWS imprinted region

-Methylated on mat chromosome

-Unmethylated on pat chromosome

2. Distinct primers identify methylated (mat) vs unmethylated (pat) alleles

Lack of mat allele =

Angelman

Lack of pat allele =

PWS

Methylation-Specific PCR in Angelman

Syndrome- Limitation

Cannot identify underlying cause of AS

Multiplex Ligation-Dependent Probe

Amplification (MLPA)- Detection

Detects copy # alternations and methylation status

MLPA- Resolution

Exon-scale copy # alterations

MPLA- Probes and process

Anneal adjacently to the target sequence

-Probes are ligated together

-Primers attached to the probes are used to amplify hybridized sequence

-Frags run through capillary gel electrophoresis

-Relative fluorescence of amplicons analyzed to identify dels/dups

Methylation Sensitive MLPA

Treat sample w/ methylation sensitive endonuclease Hhal

Methylation Sensitive MLPA- Methylated

-Hhal cannot digest

-Probe amplifies normally

Methylation Sensitive MLPA- Unmethylated

-Hhal wil digest at restriction site

-MLPA cannot amplify across cleaved DNA

What's the a theoretical method for UPD testing?

SNP arrays

1. Can identify ROH caused by complete or segmental isodisomy

2. Can raise suspicions about UPD; must be confirmed

3. CANNOT see heterodisomy

Wha't's the preferred method for UPD testing?

Short tandem repeats (STR)

UPD Testing- When it should be considered

1. Eval a subject w/ clinical, physical, or U/S features of disorders known to be associated with UPD

2. Molecular investigation of a condition that does not follow a typical Mendelian inheritance pattern

3. Prenatal trisomy or monosomy mosaicism of a chromosome known to be associated with a UPD phenotype

UPD- Syndromes associated

-Transient neonatal diabetes mellitus

-Russel Silver

-Beckwith Wiedemann

-Temple syndrome

-Kagami Ogata syndrome

-Prader Willi

-Angelman

-Mulchandani-Bhoj-Conlin syndrome

-Pseudohypoparathyroidism type 1b