8.9.16 Lecture Flashcards
What are restriction enzymes?
Restriction enzymes cleave double stranded DNA at specific nucleotide sequences.
The nucleotide sequences at which restriction enzymes cleave are often ___.
Palindromic
Restriction enzymes can cut in two ways - describe these.
Symmetrically (creates blunt ends) and asymmetrically (creates sticky ends, which can base pair)
DNA fragments can be ___ to produce new combinations.
Ligated
___ can join DNA fragments cut with the same restriction enzymes.
Base pairing
Describe the process of creating recombinant DNA.
- Restriction enzyme cuts DNA at the recognition sequence, creating 2 sticky ends.
- DNA fragment from another source is added and fragments stick together by base pairing (annealing).
- DNA ligase covalently seals the cut sites, creating recombinant DNA.
A DNA fragment can be inserted into a bacterial ___ with DNA ligase.
Plasmid
What is a plasmid?
Double-stranded DNA molecule that is separate from and can be replicated independently of chromosomal DNA.
Describe the process of incorporating DNA into a plasmid.
- Cut open the plasmid with a restriction nuclease
- Mix the cut plasmid with the DNA fragment to be cloned. Note that this fragment is prepared with the same restriction nuclease.
- DNA ligase and ATP are added.
- The ends base pair and DNA ligase seals the nicks in the backbone with covalent bonds.
Describe the process of amplification with plasmids and bacteria.
- Insert the fragment of DNA into the plasmid vector
- The recombinant plasmid is introduced into a bacterium through transformation.
- The bacterium replicates millions of times, creating many copies of plasmid.
- Lyse the bacteria and collect the purified plasmid.
Recombinant DNA can be used to produce clinically relevant proteins. Describe how this works and give two examples.
Some plasmids contain the appropriate genetic elements to force transcription and translation of the inserted DNA fragment. Cells can be forced to produce large quantities of proteins. Examples include insulin, human growth hormone, taxol, cellulose, factor VIII.
How does gel electrophoresis work?
- Cut wells in the gel.
- Submerge the gel in buffer (maintains pH, contains necessary ions)
- Apply a current
- DNA (negatively charged) runs toward the anode (positive end)
Gel electrophoresis can separate DNA by size. Smaller molecules run ___ down the gel.
Further
What type of gel is used for short DNA molecules (
Polyacrylamide gel
What type of gel is used for large DNA molecules (>50,000 base pairs)?
Pulsed-field agarose-gel electrophoresis
Complementary nucleic acid strands can bind to each other through nucleic acid ___.
Hybridization
How is DNA denatured?
Heat or high pH (alkali solution)
What is Southern blotting?
Combines electrophoresis and hybridization to detect a specific DNA molecule within a complex mixture.
How does Northern blotting differ from Southern blotting?
Northern blotting is for detecting RNA sequences
What are probes?
Single-stranded DNA molecules used to detect complementary sequences; carry radioactive or chemical markers to facilitate detection
Describe the Southern Blot analysis process.
- Cleave DNA with a restriction enzyme
- Run unlabeled DNA on agarose gel to separate by size.
- Separated nucleic acids are blotted onto nitrocellulose paper by suction of buffer through gel and paper (transferred to a membrane)
- Remove nitrocellulose paper with bound nucleic acids
- Radiolabeled probes are hybridized to separated DNA
- Labeled probes hybridized to complementary DNA bands are visualized.
What is RFLP?
Restriction Fragment Length Polymorphism
Sickle-cell disease can be detected through ___.
RFLP
In sickle-cell disease, Glu mutates to Val, which destroys ____.
A restriction enzyme cut site
How can RFLP be used to determine the presence of sickle-cell mutation?
Because the restriction enzyme cut site is removed, the mutated gene will be longer than the unmutated gene.
What does in situ hybridization do?
Locates specific DNAs within cells and genes on chromosomes.
Describe the process of FISH (Fluorescent in situ hybridization).
- Denature DNA to create single-stranded DNA.
- Bind fluorescent to complementary sequences
- Allows for visualization of multiple probes simultaneously
FISH is utilized in the clinical lab for the diagnosis of ___.
Her2 positive breast cancer
What is PCR?
Polymerase Chain Reaction; process of amplifying DNA through repeated rounds of synthesis in vitro
What are the reactants in PCR?
- DNA
- Nuceteotides
- DNA polymerase
- PCR primers
- Buffers/MgCa
Describe the steps of PCR.
- Denature DNA sample to separate DNA strands (denaturation)
- Primers bind to DNA strands (annealing)
- Polymerase synthesize new DNA strands (extension)
PCR is important for detecting genetic differences between individuals and for detecting disease-causing genes. ___ can be diagnosed with PCR.
Hemophilia
Describe diagnosis of hemophilia by PCR.
Hemophilia is a sex-linked disorder (gene for Factor VIII on X-chromosome) with a mutation in intron 18, which removes a restriction enzyme cutting site. Amplification of normal gene is cleaved into 2 fragments; amplification of the mutant gene is only 1 fragment.
Describe diagnosis of DMD by PCR.
There are 9 exon regions commonly deleted in DMD. PCR primers designed to these regions give different product sizes. Running a gel with the products shows whether or not bands are missing (missing bands indicate deletion of exons).
How can PCR be used in forensic science and paternity tests to identify the source of a DNA sample and distinguish between individuals?
DNA sequences involve short tandem repeats (STRs or microsatellites) composed of 2-6 base pair sequences. These are located in non-coding regions. Because of the variability of repeats, individuals usually inherit a different number of repeats at each STR locus from their mother and from their father. Two unrelated individuals rarely contain the same pair of sequences at a given locus. PCR using primers to recognize unique sequences on either side of one locus produce a pair of bands of amplified DNA from each individual (one for the maternal STR variant and one for the paternal STR variant)
What is Reverse Transcriptase PCR used for?
Detecting RNA templates
Reverse transcriptase produces ___ from RNA.
Complementary DNA (cDNA)
Why can’t RNA be used as a PCR template?
It is single stranded and relatively unstable
Describe the process of synthesizing cDNA.
- Total mRNA extracted from tissue.
- Short oligonucleotide complementary to the poly-A tail at the 3’ end of the mRNA (poly-T primer) hybridized to the RNA to act as a primer for reverse transcriptase.
- Reverse trasncriptase copies the RNA into a cDNA chain, forming a DNA-RNA hybrid helix.
- Degrade RNA with RNAse H (specialized nuclease) that attacks only RNA.
- DNA polymerase copies the remaining single-stranded cDNA into double-stranded cDNA.
Note that because DNA polymerase can synthesize through the bound RNA molecules, the RNA fragment that is base paired to the 3’ end of the first strand usually acts as a primer for the second strand.
Reverse-transcriptase PCR can be used to diagnose ___.
HIV
What is used to measure the expression of many genes at the same time?
Microarrays
Describe the process of using a microarray.
- Isolate mRNA from a tissue sample.
- Reverse transcribe mRNA to cDNA using fluorescently labeled nucleotides.
- Apply cDNA to microarray, which hybridizes with any complementary DNA on the array. A microarray bears copies of single-stranded DNA fragments representing an organisms genes.
- Wash to remove unbound cDNA. Scan for fluorescence. Each spot represents a gene expressed in the tissue sample.
DNA microarrays can be used in the clinic to measures mRNA expression levels for 21 genes in patients with ___.
Estrogen receptor-positive, node-negative breast cancer
In traditional sequencing, what happens?
A complementary strand of DNA is synthesized from a single strand DNA template.
Describe Sanger sequencing.
- Denature the DNA into separate strands.
- Primers bind to DNA strands.
- Polymerase synthesizes new DNA using labeled dideoxyribonucleotides (dNTPs). These are readily incorporated into a growing chain, but act as chain terminators after incorporation.
- A terminator will be inserted at every base throughout the sequence.
- The sequencing reaction is run on a PAGE gel to separate the fragments based on size.
Note that the results give the sequence of the complementary strand, not the template strand.
DNA sequencing can be used to diagnose genetic diseases caused by changes in DNA sequences, such as ___.
Cystic fibrosis
CF is commonly caused by a three-nucleotide deletion in the gene for ___.
CFTR (Cystic fibrosis transmembrane regulator, a chloride ion transporter)
Compare traditional sequencing and NGS (next generation sequencing)
- NGS is faster because the chemical reaction is combined with signal detection and it generates data from thousands of reaction simultaneously.
- NGS is more accurate because it relies on reading short overlapping fragments, leading to multiple sequencing. NGS has a greater depth of coverage.
- NGS requires less DNA.
- NGS is more cost effective in the long run.
