Tools for Molecular Biology Learning objectives Flashcards
- Recall the classes of enzymes that are used in recombinant technology to: a) copy a DNA sequence into a DNA sequence, b) copy an RNA sequence into a DNA sequence, and c) join DNA fragments
-DNA polymeraze copies a DNA into a DNA sequence
-Reverse Transcriptase copies RNA sequence into DNA sequence
DNA Ligase joins DNA fragments
2- Describe the three main stages that are repeated during PCR amplification. State the approximate temperature of each step, and relate this temperature to the state of the DNA molecules in the PCR reaction.
DNA amplification by the Polymerase Chain Reaction (PCR):
Primers are synthesized that are complementary to both strands of a specific sequence of DNA.
A thermally stable DNA polymerase (such as Taq) and the four nucleotide triphosphates are added to the reaction containing primers and DNA template. DNA strands are denatured by heating to ~95 C̊ and the solution cooled to ~55 C̊ to allow the primers to hybridize to the template.
The temperature is raised to ~72 ̊C and the polymerase uses each strand as a template and makes a copy by extending the 3’ end of the primer.
Repeat the process. Each temperature is cycle is 94 to 55 to 72 ̊C.
Repeating this cycle N times gives 2N copies, e.g. after 35 cycles of PCR, you have 235 or ~10 billion copies of the DNA of interest.
- Describe at least 1 distinct use for PCR amplification in the diagnosis of a genetic condition in your patients.
The PCR method is useful in situations in which the amount of DNA sample is limited, such as in forensics and prenatal testing, or in which the quality of the DNA sample is poor.
- Compare and contrast the molecular details of the processes of DNA sequencing and PCR amplification on in a short paragraph.
DNA Sequencing
Used when trying to identify a mutated recombinant clone.
The normal substrates for DNA synthesis are nucleoside triphosphates that are based on the sugar 2 ́-deoxyribose (dNTP).
When 2 ́,3 ́-dideoxyribose (ddNTP) is incorporated into the DNA backbone, DNA synthesis is terminated because there is no free 3 ́-OH for extension.
A primer sequence is determined
for an unknown fragment of
single-stranded DNA. This
unknown DNA is combined with
DNA polymerase, primer, dNTPs
and ddNTPs in four separate
reactions containing either a T, A,
,G or C ddNTP for manual
sequencing. Alternatively, all four
ddNTPs that have each been marked with a different fluorescent dye can be mixed for automated sequencing.
DNA synthesis is allowed to continue until many different fragments of varying lengths are produced. These fragments are separated by gel electrophoresis and visualized (by autoradiography or fluorescence detection).
The terminating ddNTP is identified by the reaction (for manual sequencing) or its color (automated sequencing), and the sequence can be “read” from the bottom of the gel to the top.
The sequence of the newly synthesized DNA (which is deduced from the gel) is the complement of the unknown strand.
DNA amplification by the Polymerase Chain Reaction (PCR):
Primers are synthesized that are complementary to both strands of a specific sequence of DNA.
A thermally stable DNA polymerase (such as Taq) and the four nucleotide triphosphates are added to the reaction containing primers and DNA template. DNA strands are denatured by heating to ~95 C̊ and the solution cooled to ~55 C̊ to allow the primers to hybridize to the template.
The temperature is raised to ~72 ̊C and the polymerase uses each strand as a template and makes a copy by extending the 3’ end of the primer.
Repeat the process. Each temperature is cycle is 94 to 55 to 72 ̊C.
Repeating this cycle N times gives 2N copies, e.g. after 35 cycles of PCR, you
have 235 or ~10 billion copies of the DNA of interest.
- Describe how DNA detection during quantitative (“real-time”) PCR is accomplished.
Quantitative PCR (“real-time PCR”). Quantitative (or “real-time”) PCR can be used to measure the abundance of specific DNA amplicons during PCR. PCR is done in the presence of a dye (SYBR green) whose fluorescence is significantly increased when it is bound to double-stranded DNA. Dye fluorescence is measured after the extension phase of PCR, when specific double-stranded products should form. This signal can be measured at each cycle, generating a diagnostic trace that indicates the accumulation of double-stranded DNA over the course of the PCR reaction. The abundance of specific templates in multiple samples can be compared using quantitative PCR
- Be aware of the different types of cloning vectors and their general features.
Other vectors for DNA cloning –
Plasmids are the vector of choice for amplification of DNA sequences in bacteria, and plasmids can also be introduced into cells of higher eukaryotes (but will not replicate in these organisms).
Bacteriophage are used to infect E. coli when high efficiency of introduction of the recombinant vector is required (take up to a 20kb insert).
Cosmids are a hybrid between plasmids and bacteriophage: they use a plasmid replication origin and can carry up to 45kb of foreign DNA, but unlike bacteriophage they do not kill their host cell.
Viral and artificial chromosomal vectors allow introduction of DNA into eukaryotic cells and can carry very large DNA inserts. Viral vectors, such as retroviruses, are used where integration into the host genome is required such as during gene therapy.
- Describe the use of microarrays for measuring levels of mRNA (e.g. gene expression).
Microarrays are ordered arrays of oligonucleotides that hybridize to RNA or DNA targets of interest. Samples are fluorescently labeled, hybridized to the microarray, and the level of RNA or DNA in the sample is inferred from the signal intensity. The identity of the probe is inferred by its position on the array, e.g. the top left probe might interrogate Gene X and the bottom right probe, Gene Y. This way, many thousands of microscopic spots can be interrogated.
- Write out three palindromic double‐stranded DNA sequences that are likely to be cut by restriction endonucleases (a known restriction enzyme does not have to exist for your sequences).
- Explain the principle of electrophoretic separation of DNA to a premed student.
Gel electrophoresis – Used to separate DNA molecules on the basis of their size. DNA has a negative charge, and in an electric field migrates towards a positive electrode. The rate of migration of a piece of DNA through a gel is inversely proportional to its size.
- Give an example of a disease that can be diagnosed using a restriction fragment length polymorphism (RFLP) and a use of DNA fingerprinting. Describe at least three experimental stages required in each of these procedures
Screening for disease alleles characterized by restriction fragment length polymorphisms (RFLP) –Disease allele causes a loss or gain of a restriction endonuclease cleavage site. A classic example is screening for the HbS mutation of sickle cell anemia that destroys a restriction site for the restriction endonuclease MstII. A patient’s DNA is digested with a “diagnostic” restriction enzyme, followed by southern or PCR analysis, then electrophoresis and detection of altered size of restriction fragment.
Paternity testing - by DNA fingerprinting of variable number tandem repeats (VNTRs). Consists of PCR or restriction digestion (followed by southern blotting) of region containing VNTRs, then electrophoresis and detection of altered size of DNA fragment patterns.
- List the names given to the transfer of DNA, RNA and protein respectively from an electrophoresis gel to a membrane
Southern blotting – Used to identify a specific DNA fragment of known sequence from a complex mixture of DNA fragments. DNA fragments are denatured and separated by gel electrophoresis. Fragments are transferred (“blotted”) onto a sheet of nitrocellulose membrane. The membrane is incubated with a radioactive DNA probe that binds to the complementary DNA sequence. Autoradiography used to detect radioactive fragments.
Northern blotting – Similar to Southern blotting, and is used to identify RNA fragments of known sequence form a complex mixture of RNA fragments.
Western Blotting for protein.
- Describe three characteristics of a hybridization probe that you will use to detect a specific DNA sequence on a membrane.
It needs to have complementary to the DNA sequence to be probed. It has to labeled (dye or radioactive).
