Chapter 3.2 Flashcards
Laboratory Techniques and Application
of Recombinant DNA Technology (6)
- Agarose gel electrophoresis
- DNA Sequencing
- Next Generation Sequencing (NGS)
- Fluorescence In Situ Hybridization (FISH)
- Southern Blotting/Southern
analysis/Southern hybridization - Northern Blot Analysis
allows one to separate and visualize DNA
fragments based on size
Agarose gel electrophoresis
high percentage of agarose= ___ dna fragments
low percentage of agarose= ___dna fragments
high percentage of agarose= small dna fragments
low percentage of agarose= large dna fragments
- a technique of
determining the nucleotide sequence of the
gene– the exact order of the bases in the
genome or gene of the organism
DNA Sequencing
Benefits of knowing the exact sequence of the gene (5)
- deduce the amino acid sequence of a protein encoded by a cloned gene
- determine the exact structure of gene
- identify regulatory elements
- identify differences in genes created by gene splicing
- identify genetic mutations
designed to produce highly accurate and long stretches of DNA sequence, greater than1 giga base (billion bases) of DNA per reaction, at a low cost
Next Generation Sequencing (NGS)
can be used to identify which chromosome
contains a gene of interest- can also be used to determine the cell type that
is expressing a particular mRNA
Fluorescence In Situ Hybridization (FISH)
steps in Next generation sequencing (4)
- DNA extraction
- Library separation
- Sequencing
- Analysis
molecular biology technique for identification
and quantification of DNA
begins by digesting chromosomal DNA into
small fragments with restriction enzymes
Southern Blotting/Southern
analysis/Southern hybridization
____
following electrophoresis, the gel is treated
with an alkaline solution to denature the DNA;
then the fragments are transferred onto a
nylon or nitrocellulose membrane using a
technique called ___
Southern
Blotting/Southern
analysis/Southern hybridization; blotting
used to visualize only specific fragments
of interest
Southern
Blotting/Southern
analysis/Southern hybridization
Southern Blotting steps (6)
- DNA extraction
- DNA Fragmentation with RE
- Separate DNA (gel electrophoresis)
- Transfer and fix separated DNA molecules onto nylon membrane
- Probe DNA of interest
- Analysis
molecular biology
technique for quantification of DNA; RNAis isolated from a tissue of interest and
separated by gel electrophoresis (the RNA is
not digested with enzymes)
Northern Blot Analysis
RNA is blotted onto a nylon membrane and
then hybridized to a probe- Amounts of mRNA
produced by different tissues can be compared
and quantified via different techniques
Northern Blot Analysis
Northern Blotting steps (5)
- RNA extraction
- Separate RNA (gel electrophoresis)
- Transfer and fix separated RNA molecules to nylon membrane
- Probe RNA interest
- Visualize and document bands
performed
when only very small amount of
mRNA
is extracted from the sample; isolated
mRNAis converted into double-stranded
cDNA by the enzyme reverse transcriptase in a
process similar to the way in which cDNA for a
library is made.
Reverse transcription PCR (RT-PCR)
enables researchers to quantify
amplification
reactions
as they occur in “real time”; basic
procedure involves the use of ___ that use a laser to scan a beam
of light through the top or bottom of each PCR
tube.
Real-time PCR or quantitative PCR (qPCR); specialized thermal cyclers
– another technique
for studying gene
expression
; created with the use of a small
glass microscope slide; single-stranded DNA
molecules are attached or “spotted” onto the
slide using arrayer
Gene microarrays/DNA microarrays/gene
chip
computer-controlled high
speed robotic arm; fitted with a number of tiny pins;
fixes the DNA onto the slide at specific
locations
arrayer
- mutations can be
created in specific nucleotides of a cloned gene contained
in a vector; gene can then be
expressed in cells, which results in the translation of a mutated protein;
Gene mutagenesis studies
____
can be a very valuable way to help scientists
identify critical sequences in genes that
produce proteins involved in human Diseases.
site-directed mutagenesis
– a technique that uses
double-stranded pieces of RNA(dsRNA) to
inhibit or silence expression of genes
RNA interference
