Tools of Molecular Genetics Flashcards
What is the major aim for molecular genetics tools?
Major aim: characterize mutations that lead to genetic disease and understand how the mutations affect health
Use this information to improve diagnosis and disease management
Two obstacles that geneticists face?
What is the remedy?
Obtaining enough DNA or RNA sequence to analyze
Purification of sequence of interest
Quantity amplified by molecular cloning and PCR
What is molecular cloning?
Isolate a defined DNA sequence
Create multiple copies in vivo
Used to amplify gene containing DNA fragments
Can be used to amplify any DNA sequence
How do the requirment elements of different host differ?
Required elements differ according to host Include: Origin of replication Selectable marker Other features are desired
What are the 4 steps to molecular cloning?
DNA fragmentation with restriction endonucleases
Ligation of DNA fragments to a vector
Transfection/transformation
Screening/selection
In molecular cloning how do we isolate fragment of interest
PCR
Restriction Enzyme Digestion
DNA sonication/fracionation
chemically synthesized oligonucleotides
Following ligation, plasmid is transformed into bacteria for propagation
Bacteria are plated on selective agar
Choose bacteria of interest
molecular cloning processes i guess
What are libraries?
Collection of clones
Each carries vector with different DNA fragment
If collection is sufficiently large, will theoretically contain all sequences from original DNA source
Can screen library to find DNA fragments of interest
what are genomic libraries?
Genomic library - population of host bacteria, each carrying DNA fragment inserted into cloning vector
Represents entire genome of source
Term also applies to collection of all the vector molecules prior to host insertion
What are cDNA libraries?
cDNA library - collection of cloned cDNA fragments inserted into host cells
What is cDNA produced from
cDNA is produced from fully transcribed mRNA
why is cDNA a useful tool?
Lack introns (eukaryotic) - readily expressed
Means easy to replicate
No information on enhancers, introns and other regulatory elements
how is cDNA created?
cDNA is created from mature mRNA using reverse transcriptase
what is reverse transcriptase
reverses central dogma of biology
screening libraries
Probes are tagged for identification Probes come from many sources: Genomic or cDNA libraries PCR produced DNA fragments Chemically synthesized DNA/RNA
Geonome Databases
Library screening still important today
Genomic databases from BAC/cDNA libraries are online and searchable
Can electronically compare with sequence of interest
Methods of nucleic acid analysis
To examine RNA or DNA from a gene, must distinguish from all other fragments in sample
Use gel electrophoresis to separate by size
Nucleic acid hybridization to find molecule of interest
Southern blotting used for?
Use to check for presence of a DNA sequence in a DNA sample
Combines agarose electrophoresis, transfer methods, probe hybridization
What is process for southern blotting
Restriction endonucleases used for cleavage
DNA fragments are electrophoresed to separate by size
DNA gel placed in alkaline solution to denature the ds-DNA
Nitrocellulose membrane is place on gel
Pressure is applied - capillary action with appropriate buffer causes DNA to move from gel onto the membrane
Ion exchange interactions bind the DNA to the membrane
Membrane is exposed to hybridization probe
Single DNA fragment with specific sequence is determined
Excess probe is removed, results are visualized
Hybridization of the probe to a DNA fragment on the filter membrane indicates the fragment contains DNA sequence complementary to probe
Difficult to identify mutations
Only those that affect size of fragment
Use of Allele-Specific Oligonucleotide Probes
With some genetic diseases, same mutation affects one or a small number of bases
Can target the DNA analysis to look for specific mutation
Best probe = synthetic oligonucleotide
