Flashcards in Tools of Molecular Biology Deck (17):
What double-stranded DNA sequences that are likely to be cut by restriction endonucleases?
Restriction endonucleases – also called restriction enzymes, cut double stranded DNA at specific sequences, restriction sites, which are usually palindromes: a sequence which is the same when read from either DNA strand in the 5ʼ-3ʼ direction. i.e. TACGTA
How does electrophoretic separation of DNA work?
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 through a gel is inversely proportional to size. Then, you can visualize it by soaking the DNA in ethidium bromide and expose it to UV light.
What are three experimental stages required in RFLP and DNA fingerprinting?
Digest patient's DNA with diagnostic restriction enzyme.Do a southern blot or PCR analysis. Gel electrophoresis to detect various sized fragments.
What are the names given to the transfer of DNA, RNA and protein respectively from an electrophoresis gel to a membrane?
Southern Blotting: allows detection of specific DNA fragments from complex mixtures via hybridizationNorthern Blotting: Used to identify RNA fragments of known sequence form a complex mixture of DNA fragments, using a method very similar to southern blotting.Western Blotting: separates proteins on the basis of size, but uses an antibody to detect instead
What 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 c) join DNA fragments.
a) DNA Polymeraseb) Reverse transcriptasec) DNA Ligase
Describe three characteristics of a hybridization probe that you will use to detect a specific DNA sequence on a membrane.
The probe must be radioactive, complementary, the proper length, and in abundant supply.
How does DNA fingerprinting work?
Do PCR for variable number tandem repeats then run using gel electrophoresis.
What are the three main stages that are repeated during PCR amplification?
1. Denature DNA (high temperature)2. Anneal primer to DNA (low temperature)3. Elongate the strand (just below Tm of primer) using Taq polymerase
What is an example of a genetic condition that PCR amplification can help diagnose?
Cystic fibrosis.This disease has hundreds of attributable mutations. Generate PCR primers that bind to a mutated allele and see if the patient is expressing one of such mutations.
Compare and contrast the molecular details of the processes of DNA sequencing and PCR amplification.
Similarities: Both use primers to initiate the copying of part of a strand of DNADifferences: PCR is used on double stranded DNA, sequencing utilizes dideoxyDNA.
What are the different types of cloning vectors?
Plasmid, bacteriophage, cosmid, BAC:bacterial artificial chromosome, YAC:yeast artificial chromosome, retrovirus
What are the general features of plasmids?
accepts 20kbE. Coli hostSimple to clone, transformation is inefficient
What are the general features of a bacteriophage?
accepts 25 kbE. Coli hostInfection 1000x more efficient than transformationGood for gDNA/cDNA libraries
What are the general features of a cosmid?
accepts 45 kbE. Coli hostMixture of phage and plasmid, larger inserts due to lack of genes that produce structural components of virus particle
What are the general features of BAC?
300 kbE. Coli hostUsed for sequencing of genomes and chromosome mapping
What are the general features of YAC?
accepts 2 MbE. Coli/yeast hostUsed for sequencing of genomes and chromosome mapping