Molecular Fundamentals - Molecular & Biochemical Techniques

what does agarose gel electrophoresis allow for?

separation of DNA fragments based on size

what does agarose form?

a porous structure when polymerized

with agarose gel electrophoresis, what fragments move faster?

smaller fragments

in PCR, what is the sequence that is amplified?

DNA template

what are the components of PCR?

• DNA template

• DNA polymerase

• pool of dNTPs (dATP, dTTP, dCTP, dGTP)

• primers

• buffer (Mg²⁺)

what does PCR allows for?

amplification of a specific sequence of DNA

what do PCR diagnostic tests test for?

the presence for an infections agent (virus, bacteria, etc) based on the nucleic acids present in the infectious agent

what are the advantages of PCR based testing?

because of the amplification capabilities, a very low level of infection can be detected

in ELISA or western blot diagnostic tests, what is required for a positive result?

the presence of antibodies against the infective agent

what describes the window period?

there is a time period between the initial infection and the production of antibodies by the infected individual’s body

what can happen since the PCR diagnostics identify the infective agent itself?

a positive result can be obtained early on in the infection process

what do restriction enzymes (restriction endonuclease) do?

cut DNA at specific nucleotide sequences by cleaving the phosphodiester bond

most restriction enzyme cleavage events leave what?

“sticky ends”

what describes the “sticky ends” produced by restriction enzyme?

single stranded at the ends, with one strand overhaning the other

since the sticky ends are complimentary, what happens?

they can be joined back together (ligated)

what describes restriction fragment length polymorphisms?

a mutation can occur in a recognition site for a restriction enzyme

the DNA from an individual with a restriction fragment length polymorphism (RFLP) mutation cannot be what?

cut at that particular site by that enzyme

DNA fragments generated from restriction fragment length polymorphism enzyme’s cut will be what?

a different length from those of a individual that does not have the mutation

RFLP technique can be used as what?

a diagnostic indicator for the presence of some genetic diseases

restriction fragment length polymorphisms use what technique?

southern blotting technique

what is a known disease associated with restriction fragment length polymorphisms?

sickle cell anemia

what describes DNA fingerprinting?

highly variable regions, regions of genomic DNA that are repeated in tandem a variable number of times

what is associated with DNA fingerprinting?

Variable Number of Tandem Repeats (VNTR)

only monozygotic (identical) twins will have what?

identical DNA fingerprints

the molecules that formed by the joining of two sticky ends is new DNA molecule called what?

recombinant DNA

in what technique, is the DNA of interest joined to a vector (carrier DNA in the form of a plasmid) creating a hybrid molecule?

DNA cloning

in DNA cloning, after the hybrid molecule is replicated by the bacterial cells and amplified, what could happen?

• the plasmid can then be purified from the bacteria

• the bacteria can be induced to make protein from the DNA that was cloned into the vector

what allows for visualization of cell structures in living things by connecting to a protein of interest?

green fluorescent protein (GFP)

what is one example of how GFP can be used?

when the sticky end of α-tublin is combined with sticky end of a plasmid containing GFP, everywhere α-tublin is expressed, GFP will also be expressed and be visible via microscope as fluorescent

what was one of the first therapeutic recombinant proteins made?

insulin

DNA cloning can also be used to synthesize what?

therapeutic proteins

what are some therapeutic proteins that have been made via DNA cloning?

• Factor VIII: used to treat hemophilia

• Tissue Plasminogen Activator (TPA): dissolves blood clots (stroke)

• Erythropoeitin: used to treat anemias

what are nucleic acid probes?

short fragments of single-stranded DNA or RNA that are used to recognize a specific sequence in the target DNA

nucleic acid probes are labeled with what?

either a fluorescent tag or by incorporation of a radioactive nucleotide

what is Fluorescence In Situ Hybridization (FISH) used for?

to detect the presence/absence of specific sequences of DNA in chromosomes (ex: activating mutations in proto-oncogenes, loss of tumor suppressor genes)

in what technique are single-stranded DNA probes (that are conjugated to fluorescent molecules) made that recognize a sequence of interest in the chromosome?

Fluorescence In Situ Hybridization (FISH)

what is the method for detecting specific sequences of DNA?

souther blot

what is the method for detecting specific sequences of RNA?

northern blot

in both northern blot and southern blot, a probe is made that has what?

a sequence complimentary to the sequence of interest in the DNA/RNA sample and is labeled with either fluorescence or incorporation of radioactive nucleotides

in both southern blot and northern blot, once the probe is incubated with the membrane containing the transferred DNA/RNA samples, what happens?

the membrane is exposed to autoradiography film and DNA/RNA fragments bound by labeled probe are visible

southern blot can be used to identify what?

genetic diseases with known mutations

what is a genetic disease that can be identified by southern blot?

sickle cell

sickle cell is caused by a point mutation in the β globin gene, which causes what?

eliminates a recognization size for the restriction enzyme Mstll

• normal β globin gene has three Mstll sites

• sickel cell β globin gene has two Mstll sites

in southern blot, the probe will be hybridized to the region between Mstll sites, so how do not mutated and mutated genes appear regarding sickle cell?

• if gene is mutated, one large band will be produced (because there is only one region between two sites)

• if gene is not mutated, two smaller bands will be produced (because there are two separate regions between the three sites)

the sanger method is a method of what?

chain-termination sequencing

sanger method is an amplification of a target DNA sequence in what?

the presence of specific modified nucleotides (dideoxynucleotides, ddNTPs)

what happens when DNA polymerase incorporates a ddNTP?

the elongation is terminated because the ddNTP does not have the 3’ -OH needed to incorporate the next nucleotide

for the sanger method, how are the products that are separated by agarose gel electrophoresis read?

the sequence is read from bottom of the gel to the top (smallest DNA fragments to the largest)

what describes polyacrylamide gel electrophoresis (PAGE)?

allows for separation of proteins based on size

what is western blot?

a technique used to detect specific proteins in a sample

in western blot technique, the proteins from the gel are transferred to a membrane. this transfer produces what?

a direct copy of the gene and the proteins will be in the same location on the membrane as they were in the gel

with western blot technique, what happens with the membrane containing the transferred proteins?

it is incubated with primary antibodies that are specific for the protein of interest and followed by incubation with secondary antibodies that are conjugated to an enzyme 

with western blot technique, once a chemiluminescent enzyme substrate is added, what happens?

a signal will be seen where the target protein is present on the membrane (gel)

what is a diagnostic testing that uses the western blot technique?

HIV antibody test

how is western blot used as HIV antibody testing?

• known virus proteins are separated on a polyacrylamide gel and the patient’s blood is used to perform a western blot

• if the patients is HIV positive, their blood will have antibodies that recognize viral proteins

when an individual is infected with the HIV virus, their body will make what?

antibodies against the viral proteins

what is ELISA (enzyme-linked immunosorbant assay)?

a very sensitive method for detecting the presence of a molecule (antibody or protein)

what are the steps of ELISA ( (enzyme-linked immunosorbant assay)?

1. antigen of interest is bound to the bottom of a plate

2. patient’s sample (blood sample containing putative primary antibodies against antigen) is added to the plate

3. secondary antibody conjugated to enzyme is added

4. colorimetric or chemiluminescent substrate is added 

5. signal (color or chemiluminescence) indicates that antigen of interest is present in patient

what is ELISA commonly used for?

to test donated blood

ELISA can be used to diagnose what?

HIV, but a false positive could occur (due to the fact that an antibody could be made not specifically for HIV antigen but can for some reason bid to it)

due to ELISA’s potential for false positives for HIV, what must be performed?

a follow-up western blot to confirm diagnosis

what is southwestern blot?

technique used to identify protein-DNA interactions

what describes southwestern blot?

• double stranded DNA probes of specific sequence are added to blot

• if the probes bind to a protein on the blot, that means that protein can bind the specific DNA sequence found in that probe

what describes microarray (also known as DNA chips or gene chips)?

single stranded DNA molecules representing all expressed genes in a cell are fixed to a solid substrate (glass slide or silicon film) and used as a reference or standard

what is microarray used for?

to compare gene expression patterns between cells exposed to two different conditions or between a healthy cell and a diseased cell

what describes the steps of microarray?

• isolate mRNA from the two samples

• use reverse transcriptase to make complimentary DNA of the mRNA (cDNA) that is labeled with a dye

• the labeled cDNAs are then hybridized to the reference DNA chip (color visible will show how much the specific gene was expressed)

what describes proteomics?

a technique used to compare the differences in expression of proteins between two samples

what describes steps of proteomics?

• proteins from two different samples (cell types) are isolated and each sample is labeled with a different fluorescent dye

• proteins are separated by 2D gel electrophoresis (first separated based on charge and then separated based on size)

• a computer program aligns the spots and determines which proteins are upregulated or downregulated based on the intensity of the spot

gene therapy is a method for what?

introducing normal copies of defective genes

gene therapy does not do what?

replace the defective gene, just adds a normal copy of the gene into the genome

for gene therapy, what happens to the normal gene?

it is incorporated into a viral particle and the virus is used to infect the patient’s cell

what describes the use of retroviruses for gene therapy?

RNA viruses that use reverse transcriptase to make a double stranded DNA copy of their RNA genome

retroviruses used for gene therapy can do what?

stably integrate into the cell’s nuclear DNA (making the therapy essentially permanent)

what are the two types of viral vectors for gene therapy?

retroviruses and adenoviruses

what describes the use of adenoviruses for gene therapy?

DNA viruses that do no integrate into the cell’s genome (therapy has to be repeated periodically)

transgenic technology makes it possible to do what?

produce organisms in which a specific gene has been replaced with a mutated version or in which the function of a specific gene is completely removed

knock-out mice are used for what?

to create an animal model of a human disease that is caused by a mutation of a specific gene

transgenics involves the utilization of what?

CRISPR (clustered regularly interspaced palidromic repeats)

what are the two major components of CRISPR?

• guide RNA (sequence that is complimentary to a gene or genetic region of interest)

• Cas9

what is Cas9?

an endonuclease capable of cutting phosphodiester backbone on both strands

CRISPR allows for what to occur?

the addition, removal, or replacement of specific genes