Part 1/2: Tools of Molecular Genetics Flashcards
Restriction enzymes:
Cleave DNA at specific sites.
- bacterial enzymes (nucleases)
- can cleave a DNA molecule at any site where a specific short sequence of nucleotides occurs (palindrome)
- Different restriction nucleases cut at different sequences.
- Extensively used in recombinant DNA technology.
What types of DNA sequences do restriction enzymes cleave?
Palindromic
(display rotational symmetry)
The two ways restriction enzymes can cleave palindromic sequences:
- blunt ends (cut through center of sequence)
- Sticky ends (uneven cuts)
DNA/RNA Gel electrophoresis:
Separates DNA/RNA fragments by size.
- smaller strands move further down the gel
- visualized with ethidium bromide (flouresces) or autoradiography
Hybridization:
Detects specific nucleotide sequences (DNA/RNA).
- Experimental process in which 2 complementary nucleic acid strands form a duplex
- uses DNA probe
- DNA/RNA double strand first denatured using high pH or temperature; denaturant removed to allow re-annealing
- “stringency of conditions”
Hybridization: how do you raise the stringency of re-annealing?
- high stringency hybridization
- increase the temperature or concentration of denaturing agent (will allow more perfect probe/strand annealing to occur)
- use shorter probes
Hybridization: effect of reduced stringency
- lower temperature and pH
- allows cross-species analyses and identification of distantly related members of a gene family
- use longer probe
Specimen type analyzed via Southern Blots:
DNA
Specimen type analyzed via Northern Blots:
RNA
Specimen type analyzed via Western Blots:
Protein
Restriction fragment length polymorphism (RFLP) steps:
Detects when the length of a detected fragment varies between individuals.
- DNA fragmented by a restriction enzyme.
- DNA fragments separated by length via gel electrophoresis.
- DNA on gel transferred to a membrane (nitrocellulose/nylon paper).
- Nitrocellulose/nylon paper with DNA fragments exposed to buffer with radioactive DNA probe.
- Paper washed, unlabeled DNA washes off.
- Autoradiography demonstrates DNA that binded to probe.
An RFLP occurs when:
- the length of a detected fragment varies between individuals.
- can be used in genetic analyses
Northern Blotting:
Determines gene expression.
- used to determine the molecular weight of an mRNA and to measure relative amounts of the mRNA present in different samples.
Northern Blotting Steps:
- run RNA fragments on gel to separate by length
- transfer to paper membrane using UV light
- hybridize membrane with radioactive probe
DNA Microarray:
Anayzes expression of many genes at once.
- A glass slide upon which a large number of short DNA molecules (typically in the tens and thousands) have been immobilized in an orderly pattern.
- Each of these DNA fragments acts as a probe for a specific gene.
DNA Microarray Steps:
- mRNA from sample obtained.
- mRNA transcribed to cDNA via reverse transcriptase.
- Above samples put on glass slide upon which a large number of short DNA molecules (typically in the tens and thousands) have been immobilized in an orderly pattern.
- Hybridization occurs.
- Gene expression/overlap determined via flourescent microscope.
Allele Specific Oligonucleotides (ASOs):
Detects a specific nucleotide sequence (allele).
- used to detect sickle cell and thallassemia mutations.
PCR steps:
- Denature DNA (heat)
- Primer Annealing (cooling - hybridize)
- Extension (DNA polymerase/free nucleotides)
- Repeat
NEEDS HEAT-STABLE DNA POLYMERASE
Polymerase chain reaction (PCR):
Amplifies specific regions of DNA.
- Technique for amplifying specific regions of DNA by a brief heat treatment to separate complementary DNA strands.
- Can amplify trace amounts of DNA.
Allele Specific PCR:
Amplifies allele-specific DNA sequences.
- uses allele specific primers that perfectly basepair to the 3’ end nucleotide of a sequence
Primers:
- small synthetic segments of single-stranded DNA
- bind to a specific region on either side of the target DNA sequence and initiates replication of the target DNA at that point.
- specify the DNA sequence to be amplified
Reverse Trancriptase PCR (RT-PCR)
Used to reverse-transcribe and amplify RNA to cDNA. Detects gene expression.
- single stranded mRNA obtained from tissue
- reverse transriptase transcribes mRNA to cDNA
- RNAase removes RNA primer
- PCR carried out
- denature
- anneal primer
- extend
- repeat
What is required for Sanger Sequencing?
- single-stranded DNA template
- DNA primer
- DNA polymerase
- normal deoxynucleosidetriphosphates (dNTPs),
- modified di-deoxynucleosidetriphosphates (ddNTPs)
- these terminate DNA strand elongation
Sanger Sequencing Steps:
Used to sequence DNA in a stepwise fashion.
- four individual reactions
- target DNA strand replicated in presence of primer, polymerase, regular dNTPs, and one of the ddNTPs (A, G, T, or C).
- Ran on a denaturing gel, separates fragments varying by one nucleotide.
- Gel read from bottom (5’) to the top (3’). Determines DNA sequence.
On a denaturing gel ran following Sanger Sequencing, what is the DNA sequence from the bottom of the gel to the top of the gel?
5’ → 3’
Automated DNA sequencing:
Used to determine DNA sequence.
- a single reaction tube with target DNA, primer, dNTPs, and all four ddNTPs (A, G, C, and T).
- Each ddNTP is labeled with a different color flourescent.
- Separated by capillary gel electrophoresis.
- Laser detects flourescence, chromatogram generated by computer.
- READ LEFT TO RIGHT
Sanger Seguencing gels are read from:
- bottom to top (5’ → 3’)
Automated DNA sequencing chromatograms are read from:
- left to right (5’ → 3’)
Complementary DNA (cDNA):
- DNA molecules made as a copy of mRNA and therefore lacking the introns that are present in the genomic DNA.
- Used to determine the amino acid sequence of a protein by DNA sequencing or to make the protein in large quantities by cloning followed by expression.
Dideoxy-DNA sequencing:
- The standard method of DNA sequencing.
- utilizes DNA polymerase and chain terminating nucleotides.
- missing 3’ OH group
Probe:
- A labeled segment of DNA or RNA used to find a specific sequence of nucleotides in a DNA molecule.
- May be synthesized in the laboratory, with a sequence complementary to the target DNA sequence.
