App - 1: Molecular Diagnostics Flashcards
What are nucleases? What are they used for in the body?
Molecular diagnostics
Nucleases cleave nucleic acids.
Ribonuclease (RNAase) - cleave RNA
Deoxyribonuclease (DNAase) - cleave DNA
Exonuclease - attack at the ends of nucleic acids (specific for 5’ or 3’ end)
Endonuclear - attack in the middle of the nucleic acid
many have no specific cut sites
restriction endonucleases (aka restriction enzymes) cut at specific sequences
What are restriction endonucleases?
Molecular Diagnostics
restriction enzymes cut at specific 4, 6, or 8 base pair sites called recognition sites -> these restriction site are typically symmetrical (palindromic). Shorter recognition sequences will randomly apppear more often in the genome than longer recognition sequences so they willl generate shorter restriction fragments. enzyme cuts the phosphate backbone at a specific recognition sites.
What is the mechanism of Sickle Cell Anemia?
Molecular Diagnostics
Autosomal recessive disorder. Point mutation in the beta globulin gene at the 6th position. Valine for glutamic acid. Glutamic acid’s negative charge cause ionic polymerization with Ca2+.
What are the advantages and disadvantages of Restriction endonucleases in molecular diagnostics?
Advantage: Simple, often robust assay to detect a
specific change in DNA sequence
Disadvantage: You need to know the mutation ahead
of time.
-These assays are good for known point mutations.
* Not used commonly in modern day labs due to availability of other
techniques
Explain the biochemistry of hybridization in nucleic acids and denaturation?
- DNA strands held
together by non-covalent
hydrogen bonds - Conditions that will
disrupt DNA strand
adhesions (denaturation) include high heat (melting
temperature or Tm) and alkaline conditions
(high pH)
High stringency hybridization:
Higher heat and lower
salt concentration
-heat=kinetic energy to overcome energy barriers and find a better match.
-salts help ionically stabilize +/- charges and removing them increases stringency
Low Stringency: lower heat and higher salt concentration
How can we use nucleic acid hybridization in Lab techniques?
Explain the Southern Blot technique.
Nucleic Acid examined: DNA
* Types of changes examined
* Larger rearrangements including “large” insertions (extra DNA), “large”
deletions (loss of DNA), large duplications * Restriction Fragment Length Polymorphisms/mutations
* Hybridization can occur even when a few base pairs
are mismatched, especially when the probes are large
and conditions are of low stringency
Fragile X
Are there other blotting techniques besides southern blot?
What is sanger sequencing and how is it performed?
ddNTPs + Capillary Electrophoresis.
Nucleic Acid examined: DNA
* Types of changes examined
* Single nucleotide changes
* Very small deletions and insertions (only a few nucleotides)
* Does not require the user to know what mutation they
are looking for or where it is located.
* Can only read relatively short sequences (100s of bps)
and is therefore not good for looking for large
insertions and deletions.
PCR
- Taq polymerase
- Thermostable
- Primers (a.k.a. oligonucleotides)
- Nucleotides
- Salts
- Water
- Target
What is RTPCR?
reverse transcriptase PCR
Real Time PCR
Nucleic Acid examined: DNA
* Types of changes examined: Can quantify input target
DNA sequences
* End point PCR can’t quantify because amplification is
exponential, the input target DNA is not the limiting
reagent, and therefore, the amount of product at the
completion of 35-40 cycles is generally
indistinguishable regardless of DNA input
HIV
- Causative agent of the acquired immunodeficiency
syndrome (AIDS) - RNA virus belonging to the Lentivirus family of
retroviridae - HIV infects CD3/4+ T lymphocytes and undergoes
reverse transcription into a DNA genome which
integrates into the host cell genome - New viral particles are made from the integrated
virus sequence
Why do we need HIV-1 Viral load testing?
* HIV-1 viral load is a strong predictor of the rate of
progression to AIDS independent of CD4 T-cell levels
* Viral load testing is used to monitor response to
therapy and make decisions about any necessary
changes in therapy
* Sometimes used for diagnosis in neonates and
patients that have been recently exposed
What is the difference between RTPCR and Real time PCR?
Immunoglobulin heavy chain gene rearrangement
