Methods For Gene Isolation Flashcards
It is a novel MOLECULAR TECHNIQUE involving in vitro enzymatic replication of defined DNA sequences. Used to AMPLIFY copy small segments of DNA or also called “MOLECULAR PHOTOCOPYING”
Polymerase Chain Reaction (PCR)
What are the three steps for Polymerase Chain Reaction (PCR)?
Denaturation, Annealing and Extension
The Heat strongly SEPARATE or DENATURES the DNA strands. The thermal denaturation of dsDNA at 94°C
Denaturation of dsDNA template
The temperature is DECREASED to 50-60°C which allows primers to attach to COMPLEMENTARY SEQUENCES.
Annealing of two oligonucleotide primers
The temperature is RAISED at 72-74°C just below the optimum of TAQ POLYMERASE
Polymerase extension of dsDNA molecules
The temperature of INITIAL DETANURATION?
94 °C to 98°C
The temperature of DENATURATION ANNEALING?
94 °C 5 °C below Tm 70 °C to 80 °C
The temperature of FINAL EXTENSION?
70 °C to 80 °C
The temperature of HOLD?
4 °C
Types of Modified Nucleic Acid Amplification Techniques?
- Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR)
- Nested Polymerase Chain Reaction (NPCR)
- Multiplex Polymerase Chain Reaction (MPCR)
- Digital Polymerase Chain Reaction (DPCR)
It was developed to AMPLIFY RNA TARGETS. RNA molecule via reverse transcriptase enzyme is converted to cDNA molecule and then utilized as a template sequence for following PCR reaction.
Application: DETECT RNA EXPRESSION
Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR)
It was developed to INCREASE SENSITIVITY and specificity of PCR.
Application: NON-SPECIFIC BINDINGS in products due to the amplification of unexpected primer binding sites.
Nested Polymerase Chain Reaction (NPCR)
It utilized a MULTIPLE PRIMER SET IN A SINGLE PCR reaction to produce amplicons with different sizes.
Application: the capability to examine for DIFFERENT TARGET GENE and organisms by one PCR reaction
Multiplex Polymerase Chain Reaction (MPCR)
It is accomplished by CAPTURING AND ISOLATING each individual nucleic acid molecule present in a sample within many chambers, zones, or regions that can localize and concentrate the amplification product to detectable levels.
Application: Evaluating the QUANTITY DNA and RNA molecules that exist in a sample
Digital Polymerase Chain Reaction (DPCR)
There are mainly TWO TYPES of DNA ANALYSES in qPCR.
- dsDNA binding dye
- Fluorophore-linked probes for specific PCR product detection
Who is the FATHER OF ELECTROPHORESIS?
Arne Tiselius
A method of SEPARATING ELECTRICALLY CHARGED substances in a mixture and a sample of the mixture is placed on a supporting medium, to which an electrical
field is applied.
Electrophoresis
What are the THREE PARTS of ELECTROPHORESIS?
- Voltage: Power supply
- Supporting medium: Paper, cellulose acetate, agarose gel, polyacrylamide
- Buffer system: Conduct Electricity by running buffer
A TYPE OF ELECTROPHORESIS in which the supporting MEDIUM is GEL
Gel Electrophoresis
What are the TWO TYPES of GEL ELECTROPHORESIS?
- Vertical Gel Electrophoresis
- Horizontal Gel Electrophoresis
the MOLECULAR SIZE of nucleic acid is expressed in MOLECULAR WEIGHT equivalent to the number of bases/ base pairs in the molecule.
Size of the Molecule
A LINEAR DNA FRAGMENT of a given size migrates at different rates THROUGH GELS
containing different concentrations of agarose.
Agarose concentration
The SUPERCOILED CIRCULAR DNA, relaxed circular DNA, and linear DNA of the same
molecular weight will migrate at different rates through the gel
DNA conformation
The rate of migration is proportional to the VOLTAGE APPLIED ↑voltage ↑rate of migration.
Voltage applied
It is the composition and IONIC STRENGTH AFFECTS DNA mobility.
Electrophoresis Buffer
It dyes USED TO STAIN DNA in gels are usually intercalating agents.
Presence of DNA stains in the gel electrophoresis buffer
Types of AGAROSE
- Standard (high melting temperature)
- Gels @ 35-38 oC; Mets @ 90-95 oC
- DNA fragments ranging from 1kb to 25kb
- Low melting temperature
-Gels @35 oC; Melts @ 65 oC - for rapid recovery of DNA from the gel
UV TRANSILLUMINATOR to view the fluorescent DNA in the gel.
Detection of DNA
DYE AND SIZE must be the same as DNA being measured.
Quantification of DNA
The bond appears as compact, with NO DOUBLE BOND or FAINT BAND.
Evaluation of the Quality of DNA
It involves finding out the WHOLE SEQUENCE of a person’s DNA. The laboratory procedure determines the order of bases in the genome of an organism in one process.
Genome Sequencing
Scientists begin by USING MOLECULAR SCISSORS to cut the DNA, which is composed of millions of bases: A’s, C’s, T’s, and G’s, into pieces that are small enough for
the sequencing machine to read.
DNA Shearing
Scientists ADD SMALL PIECES of DNA TAGS, or BARCODES, to identify which piece of sheared DNA belongs to which bacteria. This is similar to how a bar code identifies a product at a grocery store.
DNA bar-coding
The BAR-CODED DNA from MULTIPLE BACTERIA is combined and put in the whole genome sequencer. The sequencer identifies the A’s, C’s, T’s, and G’s, or bases, that make up each bacterial sequence. The sequencer uses the bar code to keep track of which bases belong to which bacteria.
Whole genome sequencing
Scientists USE COMPUTER ANALYSIS tools to compare bacterial sequences and identify differences. The number of differences can tell the scientists how closely related
the bacteria are, and how likely it is that they are part of the same outbreak.
Data Analysis
INSERTION OF A FRAGMENT OF DNA carrying a gene into a cloning vector and subsequent
propagation of recombinant DNA molecules into many copies is known as
Gene Cloning
A sample population of PURIFIED DNA is digested with ONE OR MORE RESTRICTIONS ENDONUCLEASES, generating fragments that are several hundred to thousands of base pairs in length.
Southern-blot hybridization
The samples CONTAIN UNDIGESTED RNA instead of DNA. The principal use of this method is to obtain information on the expression patterns of specific
genes.
Northern blot hybridization
Is an IMMUNOBLOTTING TECHNIQUE that relies on the specificity of binding between a
molecule of interest and a probe to allow the detection of the molecule of interest in a
a mixture of many other similar molecules.
Western Blot
