Unit II- Molecular Technology Flashcards
1
Q
What is Molecular Biology
A
- recombinant DNA technology or molecular genetics
- a collection of methods used to isolate, manupulate, amplify, re-organize, and analyze genes at the molecular level
- methods that have revolutionized and will continue to revolutionize research, diagnostics, and practice of medicine
- made possible by learning: the structure of DNA, restriction and modification of bacteria, DNA sequencing, DNA uptake by bacteria, bacterial plasmids and cloning, therophilic bacteria, automation, minitiarization, deep sequencing
2
Q
Nucleic Acid Hybridization
A
- inherent feature of DNA/RNA: double stranded complementarity
- allows one to “search” for homologous sequences in complex mixtures and/or to discriminate between related sequences
3
Q
Restriction Enzymes
A
- part of the bacterial defense system called restriction and modification
- usually cuts dsDNA in palindromic sequences
- can cut to leave staggered ends (most useful) either with 5’ and 3’ overhands
- staggered ends can be efficiently ligated to complementary ends with T4 DNA Ligase
- can serve as landmarks on DNA sequences and therefore can be used for linkage analysis (RFLPs)
- think EcoRI
4
Q
Restriction Maps
A
- formed the basis for the 1st molecular method to perform genetic linkage analysis called RFLPs
- shows where all the restriction enzymes cut
5
Q
Sizing of nucleic acids: Gel Electrophoresis
A
- nucleic acid is separated by size through a molecular sieve (gel)
- voltage is used to move the negatively charged nucleic acids toward the anode (positive_
- two gel choices: agarose-large, acrylamide-small
- detection: Fluorescent dyes (EtBr), Radioactivity-film or phosphoimager screen, probes
6
Q
Labeling DNA to make “probes”
A
- isolate fragment to be labeled
- prime a DNA synthesis reaction with homologous oligos
- synthesize complementary strands with a DNA polymerase in the presence of radioactively labeled (32P) nucleotide precursors
7
Q
Southern Blotting
A
- separate DNA by size using gel electrophoresis
- make a paper copy of the gel: use voltage to transfer the DNA onto nitrocellulose paper, HIGH pH and/or salt denature the DNA
- hybridize a 32P-labeled DNA “probe” to the paper
- wash non-specific probe by binding off of the paper
- expose the paper to X-ray film or a phospho-imager screen
8
Q
Cloning
A
- isolate DNA fragment and vector cut to leave completementary “sticky” ends
- ligate (splice) the fragment into the vector
- transform the chimearic molecules into a host (bacteria)
- screen for the clones with the insert
9
Q
Vectors
A
- vehicles used to move, manipulate and amplify cloned genes/fragments of genes
- they are human altered versions of naturally occuring DNA elements used to transfer genetic information
- they vary in the amount of recombinant DNA they can be tricked into carrying and the hosts that can maintain them
10
Q
Bacterial plasmids
A
- based upon F1 plasmids
- small closed, dsDNA circles
- bacteria are the host, introduced by chemical transformation
- carry an origin of replication, an antibiotic resistance gene and a poly-cloning site (a short stretch of DNA that has many restriction enzyme cut sites)
- can only accept up to 15 kb of recombinant DNA
11
Q
Bacteriophage Lambda
A
- a bacterial virus with a 45 kb linear dsDNA genome
- during infection viral genes hijack the bacterium to produce millions of viral particles
- bacterial lysis spills the viral particles into the growth medium
- recombinant DNA up to 20 kb can be inserted into the middle non-essential region of the viral genome
12
Q
Cosmids
A
- gutted versions of lambda all but the ends have been removed
- can clone up to 45 kb of recombinant DNA
- recombinant genome is packaged into viral particles in a helper strain: Cos ends direct replication and packaging
13
Q
Bacterial Artificial Chromosomes (BACs)
A
- dsDNA closed circles (plasmids) that are maintained in E. coli as if they were the cell’s chromosome (low copy #)
- can accept 100-300 kb of recombinant DNA
14
Q
Yeast Artificial Chromosome (YACs)
A
- linear dsDNA recombinant molecules maintained in yeast as a chromosome
- telomeres on ends, centromere in middle and origins of of replication insure stable maintenance
- can accept 100-2,000 kb recombinant DNA
- not as good because they cross-over more
15
Q
Libraries
A
- a large collection of random inserts for a genome of interest in a cloning vector
- analogous to the Library of Congress after a large earthquake (all mixed up)
- used to “fish out” the gene or genes you are interested in studying
- ideally all parts of the genome are represented as overlapping fragments
- two types: gemonic and cDNA
16
Q
cDNA library
A
- purify mRNA from cells/tissue of interest
- affinity purify on an oligo-dT coupled column to isolate poly(A) mRNA
- prime with oligo-dT and make complementary DNA strands with Reverse Transcriptase
- Degrade the RNA strand
- Use DNA polymerase to make the second DNA strand
- ligate into a cloning vector (usually a bacterial plasmid)
- representatives in the library are only expressed genes with introns removed
17
Q
Screening a library for a gene
A
- there is a petri disk with colonies of bacteria containing recombinant plasmids
- a piece of absorbant paper is put on, and the paper is peeled from dish to produce replica of colonies
- lyse bacteria and denature DNA with alkali
- radioactively labeled DNA probe is added
- bacteria incubated with probe and washed
- colonies containing plasmid of interest were exposed to photographic film
18
Q
PCR
A
- if you know the sequence of a region of nucleic acid (RNA or DNA), PCR allows you to amplify the sequence from complex mixtures of nucleic acids even if the copies of that sequence are inabundant
- requires 2 primers, dNTPs, starting sample that contains the first template, a thermophilic polymerase
- bionomial expansion: amount of DNA made= # of starting templates x 2^#cycles. Product analyzed by gel electrophoresis
