Molecular Testing Flashcards
Exam 4
What is reverse transcription?
RNA is converted back into DNA. This can be used to create DNA copies of an RNA gene that is being studied.
What is recombinant DNA?
When DNA from one organism is injected into a carrier DNA molecule or vector (like a plasmid) which can then be introduced into a smaller host organism for study. It can be used to quickly produce clones of a gene.
What tools can be used for molecular cloning?
Vectors, gel electrophoresis, restriction endonucleases
What is a vector and what is the most commonly used vector?
A vector is a DNA molecule of known nucleotide sequence that is used to carry a foreign DNA fragment into a host organism. The most common vector used is a plasmid.
What is gel electrophoresis and how is it used in relation to vectors?
Gel electrophoresis is a method used to separate DNA fragments based on their electrical charge and size. It can be used to isolate and purify a vector.
What is a restriction endonuclease?
It is an enzyme that cleaves DNA at a specific sequence in a predictable fashion so it can be pasted into a new fragment.
What is restriction enzyme mapping and restriction fragment length polymorphisms (RFLPs)?
Restriction enzyme mapping is figuring out the distance in given sequences of DNA between points recognized by restriction endonucleases. This creates what is called restriction fragment length polymorphisms (RFLP) which can be used to identify different genotypes.
What is a recombinant protein?
A recombinant protein is the protein that is expressed from a cloned gene (or recombinant DNA)
List some recombinant proteins that are used in transfusion medicine.
Interferon-α to treat hairy cell leukemia, hepatitis B and C, hepatitis B vaccine, recombinant anti-hemophiliac factor, granulocyte colony stimulating factor (GCSF) to increase production of hematopoietic stem cells for stem cell transplant.
What is polymerase chain reaction (PCR)?
It is an alternative to cloning for isolating large amounts of single DNA fragments or genes. It is completed in-vitro and can amplify the target DNA sequence several million times.
Explain the materials needed and steps in PCR.
To complete PCR, two primers for a target region, DNA polymerase, 4 dNTPs, and magnesium are needed. Step 1 is heat denaturation. This involves applying hot temperature to break hydrogen bonds between complementary nucleotides to split the two strands of DNA. Step 2 is to anneal primers to target sequences flanking the fragment of interest by cooling to 50-60C which promotes reestablishment of hydrogen bonds. Step 3 is primer extension by DNA polymerase done at 72C. The polymerase incorporates nucleotides to the 3’ end of primers using the target DNA as the template. The cycle is completed by constantly cycling through the different temperatures. The rest of the original DNA does not get amplified due to the lack of primers.
Describe the process of DNA sequencing.
DNA sequencing is used to determine the exact nucleotide sequence by either molecular cloning or PCR. What is different is that instead of just dNTPs, a small amount of modified dNTPs lacking the hydroxyl group are incorporated (called ddNTPs). When the polymerase selects a ddNTP to attach to the sequence the chain is terminated because no new phosphodiester linkages for the following nucleotide are possible. These four ddNTPs are also labeled with a fluorescent dye (a different color for each of the four nucleotides). There will be many differently sized DNA fragments based on when the ddNTP was incorporated and all fluorescently labeled based on the last nucleotide added. These fragments can then be separated by size using gel electrophoresis and can be placed in order to determine the nucleotide sequence.
What is the difference between a dNTP and ddNTP?
dNTPs are normal nucleotides. ddNTPs lack a hydroxyl group causing the chain to be terminated and are fluorescently labeled based on the type of nucleotide.
What methods can be used to detect nucleic acids and proteins?
Nucleic acid hybridization, PCR based techniques, and western blot
What is nucleic acid hybridization?
The process of renaturation of RNA or DNA. Labeled DNA (with either radioactive, florescent, or chemiluminescent labels) is used as a probe. Whether or not the sequences renature can be detected using imaging.
What techniques use nucleic acid hybridization?
Southern blotting, northern blotting, DNA microarrays, Fluorescent in situ hybridization (FISH)
What is real time PCR?
The process is the same as normal PCR, except the product formed during each cycle of amplification is detected by fluorescence at the same time it’s produced. The DNA probes are labeled with fluorphores which emit fluorescent light when binding.
What is reverse transcriptase PCR?
This is used to detect single copies of RNA. Before PCR amplification, a step of reverse transcription is added to synthesize cDNA. Then PCR is carried out normally.
What viruses is reverse transcriptase PCR good at detecting early on?
HIV, Hepatitis B, Hepatitis C
What is transcription mediated amplification (TMA)?
This is a type of amplification where RNA is the template and the RNA molecules are amplified. No DNA is ever amplified in the process. You begin with an RNA template and use reverse transcriptase to synthesize a DNA/RNA hybrid. RNase H removes RNA from the hybrid leaving only the cDNA. A second primer binds to create a completed dsDNA. RNA polymerase will then bind and transcribe the DNA to RNA. This makes many copies of the RNA. The cycle then begins again to be repeated many times.
What is the hybridization protection assay (HPA)?
This is a type of transcription mediated amplification. Here the ssDNA probes have chemiluminescent labels that form the hybrids with RNA. Light is emitted when hybridization occurs allowing us to detect RNA present in the sample.
What is nucleic acid testing (NAT)?
This is a standard method used in blood banking to detect pathogens (such as HIV, Hepatitis B and C, and West Nile Virus) before the immune response. It uses amplification through transcription mediated amplification and detection through hybridization protection assay.
What is the Western Blot?
This is used to detect proteins by incubation with labeled antibodies or probes. The proteins are first separated with polyacrylamide gel electrophoresis and then transferred from the gel to a filter membrane. The protein is then incubated with the labeled antibody or probe which can then be detected.
What infectious disease is confirmed using the western blot for blood donations?
HIV
Describe three techniques that can be used to detect gene polymorphisms.
- Restriction fragment length polymorphism (RFLP)- this can detect glycosyl transferase genes in individuals who are AO or BO, determine if individuals are carriers for sickle cell anemia, and for HLA typing, paternity testing, and in forensic science. The nucleotide difference between the variation in these genes can cause restriction enzyme sites to be lost resulting in different sized DNA fragments. 2. PCR and allele specific probes - PCR can amplify polymorphic genes which are hybridized with probes. The probes can label different specific alleles (called specific oligonucleotide probe). This is commonly used for HLA typing. 3. DNA sequencing - This can be used for HLA typing for allogeneic hematopoietic stem cell transplants
List situation where red blood cell genotyping may be performed.
Fetal DNA typing, blood group typing of donors or alloimmunized patients, screening of blood donors to find rare phenotypes, blood group typing of patients with AIHA or other diseases
