First Aid Chapter 4 Molecular Biology Technology Flashcards
What are TRECs? Why is it useful to monitor them?
—T lymphocytes develop in the bone marrow and undergo maturation in the thymus. During T-cell receptor rearrangement, extrachromosomal DNA byproducts, called T-cell receptor excision circles (TRECs) are generated. Measurement of TRECs via PCR allows monitoring of the development of naïve T lymphocytes that have recently emigrated from the thymus. In healthy individuals, the number of TRECs declines with increasing age.
What are the disadvantages of measuring TRECs?
TREC concentration can be affected by T-lymphocyte division and T-lymphocyte death.
What are the uses of measuring TRECs?
- Diagnosis of SCID and 22q11 deletion syndromes.
- HIV-1 infection causes decreased levels of TRECs in peripheral blood T lymphocytes. Successful treatment with highly active antiretroviral therapy (HAART) increases TREC levels in peripheral blood T lymphocytes, possibly by decreasing T-lymphocyte turnover.
- Downs syndrome can present with low TREC levels in a newborn.
- Monitoring of immune reconstitution after bone marrow transplant.
How does rt-pcr work? What are the advantages of using it? What are the disadvantages? What is it used for?
Methods—RT-PCR is a sensitive method for detecting mRNA expression levels. RNA is first reverse transcribed into cDNA using a reverse transcriptase. The resulting cDNA is used as templates for subsequent PCR amplification with primers specific for one or more genes.
Advantages—Most sensitive technique for mRNA detection and quantitation. It can be used to quantify mRNA levels from much smaller samples and is sensitive enough to enable quantitation of RNA from a single cell.
Disadvantages—Accuracy is dependent on variations during amplification.
Uses—Widely used in the diagnosis of genetic diseases and commonly used in studying the genomes of viruses whose genomes are composed of RNA, such as influenza A and retroviruses like HIV.
Fig 4-26
What is In Situ Hybridization? What is Fluorescent in situ hybridization (FISH)?
Methods—A method of localizing and detecting specific mRNA sequences in morphologically preserved tissue sections or cell preparations by hybridizing the complementary strand of a nucleotide probe to the sequence of interest.
Fluorescent in situ hybridization (FISH) uses fluorescence microscopy to find where the fluorescent probe binds to the chromosomes (Figure 4-27).
What are advantages of ISH (in situ hybridization) and FISH (fluorescent in situ hibridization)?
Advantages—Does not require living cells and can be quantified automatically. Can be used to determine the structure of chromosomes. ISH can also use two or more probes, labeled with a radioactive label or the other nonradioactive labels, to simultaneously detect two or more transcripts.
What are uses of FISH? What are diseases that can be diagnosed using FISH?
- FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification.
- FISH can also be used to detect and localize specific mRNAs within tissue samples.
- Diseases that can be diagnosed using FISH include acute lymphoblastic leukemia (ALL); DiGeorge syndrome [Deletion 22q11 or velocardiofacial syndrome (VCSF)]; and Down syndrome.
TREC assay has been introduced in the newborn screens of several states for diagnosis of which disease?
SCID. The absence of TRECs in a newborn reflects T-cell lymphocytopenia. The assay can also detect 22q11 deletion syndromes and trisomy 21.
What are gene chips and how to they work? What are the advantages of using them? What are the uses?
Methods—Gene chips are glass substrate wafers containing many tiny cells, each of which holds DNA from a different human gene; the genetic material and a fluorescent probe react. Hybridization of the added nucleic acid and a piece of the tethered DNA will occur if the sequences complement one another. The development of fluorescence on the chip’s surface identifies regions of binding, and the known pattern of the tethered DNA can be used to deduce the identity of the added sample (Figure 4-28).
Advantages—Possible to carry out a very large number of genetic tests on one sample simultaneously (e.g., up to 260,000 genes can be probed on a single chip). This is useful when one wants to survey a large number of genes quickly or when the sample to be studied is small.
Uses
- To genotype or resequence mutant genomes
- To identify single-nucleotide polymorphism (SNP) among alleles
What are the two DNA sequencing methods? What are the advantages of each? What are the uses of each?
DNA Sequencing
Methods—Technique for determining the precise order of nucleotides within a DNA molecule. Several different techniques have been developed to sequence DNA.
-Developed in 1977, the Sanger method is based on the concept of the “chain-terminating” technique. It became the method of choice for its relative ease and reliability. This was the most commonly used method until the advent of next generation of techniques in early 2000.
-Second-generation techniques (Next-gen) utilize a variety of methods, including fluorescent labeling, capillary electrophoresis, and general automation. Examples include pyrosequencing, sequencing by synthesis, ion semiconductor, and sequencing by ligation. Goals of next-gen sequencing include lowering the cost of DNA sequencing while increasing productivity by replicating thousands or millions of sequences concurrently.
Advantages
- Sanger method: Simple and sensitive. Long individual reads useful for many applications.
- Next-generation methods: Variable. Overall lower cost per base in DNA sequence with potential for higher sequence yield.
Uses—Identification of specific mutations in genes associated with disease can aid in diagnosis, prognosis, and possible use of therapeutic gene therapy in the future.
For example, the location (exon) and type of mutation (loss of /gain of function) in the WAS protein determines clinical phenotype of disease.
