Tools of Molecular Genetics

Question 1

What is the major aim for molecular genetics tools?

Answer 1

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

Question 2

Two obstacles that geneticists face?

What is the remedy?

Answer 2

Obtaining enough DNA or RNA sequence to analyze

Purification of sequence of interest

Quantity amplified by molecular cloning and PCR

Question 3

What is molecular cloning?

Answer 3

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

Question 4

How do the requirment elements of different host differ?

Answer 4

Required elements differ according to host
Include:
Origin of replication
Selectable marker
Other features are desired

Question 5

What are the 4 steps to molecular cloning?

Answer 5

DNA fragmentation with restriction endonucleases
Ligation of DNA fragments to a vector
Transfection/transformation
Screening/selection

Question 6

In molecular cloning how do we isolate fragment of interest

Answer 6

PCR
Restriction Enzyme Digestion

DNA sonication/fracionation

chemically synthesized oligonucleotides

Question 7

Following ligation, plasmid is transformed into bacteria for propagation

Bacteria are plated on selective agar

Choose bacteria of interest

Answer 7

molecular cloning processes i guess

Question 8

What are libraries?

Answer 8

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

Question 9

what are genomic libraries?

Answer 9

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

Question 10

What are cDNA libraries?

Answer 10

cDNA library - collection of cloned cDNA fragments inserted into host cells

Question 11

What is cDNA produced from

Answer 11

cDNA is produced from fully transcribed mRNA

Question 12

why is cDNA a useful tool?

Answer 12

Lack introns (eukaryotic) - readily expressed

Means easy to replicate

No information on enhancers, introns and other regulatory elements

Question 13

how is cDNA created?

Answer 13

cDNA is created from mature mRNA using reverse transcriptase

Question 14

what is reverse transcriptase

Answer 14

reverses central dogma of biology

Question 15

screening libraries

Answer 15

Probes are tagged for identification
Probes come from many sources:
Genomic or cDNA libraries
PCR produced DNA fragments
Chemically synthesized DNA/RNA

Question 16

Geonome Databases

Answer 16

Library screening still important today

Genomic databases from BAC/cDNA libraries are online and searchable

Can electronically compare with sequence of interest

Question 17

Methods of nucleic acid analysis

Answer 17

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

Question 18

Southern blotting used for?

Answer 18

Use to check for presence of a DNA sequence in a DNA sample

Combines agarose electrophoresis, transfer methods, probe hybridization

Question 19

What is process for southern blotting

Answer 19

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

Question 20

Use of Allele-Specific Oligonucleotide Probes

Answer 20

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