Lab test 1 Nucleic Acids Flashcards
What is the first step in molecular diagnostic?
What temp are nucleic acids stored at?
Isolation of nucleic acids.
Nucleic acids are usually stored at 70 deg. celsius
Various methods for DNA/RNA isolation depending on the purpose of testing.
Crucial to avoid contamination of samples which leads to false results
Applications of molecular diagnostics
- Human clinical molecular diagnostic testing(germline mutations in the BRCA1 and BRCA2 genes causing hereditary breast and ovarian cancer
- Forensic and identity testing(paternity and other identity tests)
- Molecular microbiology (detection of bacteria eg, tuberculosis, legionella and viruses eg hep B, herpes simplex, influenza)
- Resistance of bacteria to antibiotics (Strep pneumonia, methicillin-resistant staph auerus)\Human leykocyte antigen(HLA) typing (histocompatility testing or immunogenetics)
In molecular diagnostics, DNA can be isolated from various biological materials:
- Human whole blood
- Human serum(to detect baceria and DNA-viruses)
- Body fluids-urine,amniotic fluid,cerebrospial fluid
- Human tissue
- Forensic evidence-semen, hair root, tissues
- Buccal swab
Blood samples should be stored in what?
Stored in EDTA vacutainer tubes.
Other anticoagulants also can affect DNA analysis
The steps of DNA isolation from blood
- lysis of the cells that do not contain nuclei(erythrocytes)
- leukocyte centrifugation to obtain pellet
- lysis of the leukocyte pellet in lysis buffer
- deproteinization by precipitation of the protein with urea, NaCL or LiCl
- centrifuging the precipitate protein
- precipitation of the DNA with an organic solven-with isopropanol
- washing the precipitate with 70% ethanol and drying
- dissolving of the DNA in the buffer
- check concentration and purity of the isolate
Where is RNA isolated from? Why is the process more complicated than DNA isolation?
Pure intact RNA is critical for what analysis?
RNA analysis is used for diagnosis of…
Gene expression is useful for…..
RNA isolated from blood, plasma serum and tissues. Process is more complicated than DNA isolation by ubiquitous presence of ribonclease enzymes in cells and tissues which can rapidly degrade RNA.
Pure intact RNA is critcal for quantitative real time PCR, RT-PCR and microarray analysis. RNA analysis is used for diagnosis of RNA -virus infection(HCV, HIV) and for gene expression(eg. moleclar profiling of breast cancer).
Gene expression analysis is useful for disease classification, diagnosis, prognosis and tailoring treatment to underlying genetic determinants of pharamacological response nd for clincal oncology
What does PCR allow?
What segment must be known?
What are needed?
Allows any DNA segment to be replicated(amplified) without the need for restricition enzymes, vectors or host cells.
Nucleotide sequence of a segment must be known
Two oligonucleotides (primers) are needed which each hybridize with one of the strands at each end of the DNA segment to be amplified. You also need enough quantites of the 4 deoxyribonucleoside (dNTP) and a heat tolerant DNA polymerase from thermostable bacteria
Describe PCR chain reaction
First, the double strand DNA is heated around 90 deg cel. to seperate the DNA double helix into single strands, the mixture is then cooled o allow hybridization of primers
Starting from primers, complementary DNA strands are now synthesized in both directions by the polymerase
Cycle is repeared 20-40 times with the same reaction mixture (cycle 2 and subsequent cycles). The cyclic heating and cooling are carried out by computer-controlled thermostats. After the 3rd cycle, double strands start to form with a length equal to the distance between 2 primers. The proportion of these approximately doubles during each cycle until all segments have the correct length
DNA electrophoresis is usually performed for what?
What three factors does the mobility of molecules in an electric field of a given strength depend on?
In proteins, all three factors vary but for nucleic acids….
When electrophoresis is carried out in a wide-meshed support material that does not seperate according to size and shape, mobility of molecules depend on….
Performed for analytical purposes, after amplication of DNA via PCR. Seperation of DNA fragments is simpler than protein electrophoresis
Mobility of molecules in an electric field of a given strength depends on size, shape and charge of molecules
For nucleic acids the ratio of mass to charge in nucleic acids is constant becuase all nucleotide components have similar masses and carry one negative charge
Mobility of molecules depend on mass alone
What gel is used for electrophoresis? Is the gel stable and how is it poured?
How do you make seperated fragments visible?
Agarose gels, a supporting material in genetic engineering, are not stable so they are poured horizontally into a plastic chamber in which they are used for seperation,
To make seperated fragments visible, after running the procedure, the gels are placed in solutions of ethidium bromide. This is an intercalator that shows strong fluorescence in UV light after binding to DNA, although it barely fluoresces in an aqueous solution
What gel for electrophoresis provides high resolution of seperation of DNA molecules 10-3000 bp long?
Polyacrylamide gels are also used in…
What does the gel consist of? What is acrylamide concentration range?
What are lower% gels better for? Higher % gels?
Polyacrylamide gel. DNA molecules varying in size by only a single base pair can be resolved under appropriate conditions.
Polyacrylamide gels are also used in DNA sequencing
The gels typically consist of acrylamide, bisacrylamide and buffer with an adjusted pH. The acrylamide concentration of the gel can also be varied, generally in the range from 3% to 20%.
Lower % gels are better for resolving very long DNA fragments, while much higher % are needed to resolve smaller DNA fragments like PCR products.
Reverse transcription polymerase chain reaction(RT-PCR) is a variant of….
Describe RT-PCR technique
Application of RT-PCR
RT-PCR is a variant of PCR
RT-PCR where a RNA strand is reverse transcribed into its DNA complement(complementary DNA, or cDNA) using the enzyme reverse transcriptase, and resultng cDNA is amplified using PCR. Do not confuse with real time polymerase chain reaction(Q-PCR/qRT-PCR)
Application of RT-PCR: In viral infection it is difficult to detrmine the species of pathogen, so RT-PCR is used to identify RNA viruses(HIV, HCV). In reverse transciptase is used to transcribe the viral RNA into dsDNA and then PCR is employed to amplify a segment of this DNA with virus specific primers. In this way, an amplificate with a characteristic length can be obtained for each pathogen and identified using gel electrophoresis.
Most genes are present in same quantity in every cell; __ copy per haploid cell and __ copies per diploid cell.
Does gene expression patterns vary from cell type to cell type, even within the same cell?
mRNA levels sometimes correlates with…. How is the quantity of individual mRNA transcripts determined?
1 copy per haploid cell and 2 copies per diploid cell. But level at which a gene is expressed as indicated by mRNA quanitites can vary widely ranging from no expression to hundreds of mRNA copies per cell.
Yes Gene expression patterns vary from cell type to cell type eg, muscle cell from nerve cell, Even within the same cell, gene expression levels may vary as the cell responds to changes in physiological circumstances.
mRNA levels sometimes correlates with levels of proteins expressed but this correlation does not always hold. The quantity of individual mRNA transcripts can be determined by quantitative PCR(qPCR) or real-time PCR.
Describe Quantitative PCR(qPCR) or real-time PCR
RNA is first isolated from the cell or tissue of interest. With the use of reverse transciptase, cDNA is prepared from this RNA sample. In one qPCR approach, the transcript of interest is PCR amplified with the appropriate primers in the presence of the dye SYBR Green I, which fluoresces brightly when bound to double stranded DNA. In the initial PCR cycles, not enough duplex is present to allow a detectable fluorescence signal. However, after repeated PCR cycles, the fluorescence intensity exceeds the detection threshold and continues to rise as the number of duplexes corresponding to the transcript of interest increases. Importantly, the cycle number at which the fluorescence becomes detectable over a defined threshold (or CT) is indirectly proportional to the number of copies of the orignal template. After the relation between the original copy number and the CT has been established with the use of a known standard, subsequent qPCR experiments can be used to determine the number of copies of any desired transcript in the orignal sample provided the appropriate primers are available,
What does Human genome contain?
What does DNA fingerprinting - PCR-STR use?
How many alleles in each STR and how much does 1 individual posseses?
Human genome contains non-coding repetitive DNA sequence, the length which varies from individual to idividual.
The method uses highly polymorphic regions that have short repeated sequences(STR) of DNA(the most common is 4 bases repeated, but there are other lengths in use, including 3 and 5 bases).
Each STR can occur in 5 to 15 different lengths (alleles) of which one individual possesses only 1 or 2.
When the various allele combinations for several STRs are determined after PCR amplification of the DNA being investigated, a “genetic fingerprint” of the individual from whom the DNA originates is obtained. Using comparative material eg, salival samples, definite identification is then possible
Each STR is polymorphic but the number of alleles are…
Each STR allele will be shared by how many individuals?
Why is STR an excellent identification tool?
Each STR is polymorphic, but the number of alleles is very small.
Typically each STR allele will be shared by around 5-20% of individuals.
Excellent identification tool: The power of STR analysis comes from looking at multiple STR loci simutaneously. The pattern of alleles can identify an individual quite accurately. The more STR regions that are tested in an individual the more discriminating the test becomes. Systems which amplify 13 core loci are almost universal .
What are the 4 steps of PCR-RFLP?
DNA isolation
Performing of PCR reaction
Digestion with restriction enzyme of PCR products
Porducts seperation on gel
