Methods For Gene Isolation

Question 1

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”

Answer 1

Polymerase Chain Reaction (PCR)

Question 2

What are the three steps for Polymerase Chain Reaction (PCR)?

Answer 2

Denaturation, Annealing and Extension

Question 3

The Heat strongly SEPARATE or DENATURES the DNA strands. The thermal denaturation of dsDNA at 94°C

Answer 3

Denaturation of dsDNA template

Question 4

The temperature is DECREASED to 50-60°C which allows primers to attach to COMPLEMENTARY SEQUENCES.

Answer 4

Annealing of two oligonucleotide primers

Question 5

The temperature is RAISED at 72-74°C just below the optimum of TAQ POLYMERASE

Answer 5

Polymerase extension of dsDNA molecules

Question 6

The temperature of INITIAL DETANURATION?

Answer 6

94 °C to 98°C

Question 7

The temperature of DENATURATION ANNEALING?

Answer 7

94 °C 5 °C below Tm 70 °C to 80 °C

Question 8

The temperature of FINAL EXTENSION?

Answer 8

70 °C to 80 °C

Question 9

The temperature of HOLD?

Answer 9

4 °C

Question 10

Types of Modified Nucleic Acid Amplification Techniques?

Answer 10

1. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR)
2. Nested Polymerase Chain Reaction (NPCR)
3. Multiplex Polymerase Chain Reaction (MPCR)
4. Digital Polymerase Chain Reaction (DPCR)

Question 11

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

Answer 11

Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR)

Question 12

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.

Answer 12

Nested Polymerase Chain Reaction (NPCR)

Question 13

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

Answer 13

Multiplex Polymerase Chain Reaction (MPCR)

Question 14

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

Answer 14

Digital Polymerase Chain Reaction (DPCR)

Question 15

There are mainly TWO TYPES of DNA ANALYSES in qPCR.

Answer 15

1. dsDNA binding dye
2. Fluorophore-linked probes for specific PCR product detection

Question 16

Who is the FATHER OF ELECTROPHORESIS?

Answer 16

Arne Tiselius

Question 17

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.

Answer 17

Electrophoresis

Question 18

What are the THREE PARTS of ELECTROPHORESIS?

Answer 18

1. Voltage: Power supply
2. Supporting medium: Paper, cellulose acetate, agarose gel, polyacrylamide
3. Buffer system: Conduct Electricity by running buffer

Question 19

A TYPE OF ELECTROPHORESIS in which the supporting MEDIUM is GEL

Answer 19

Gel Electrophoresis

Question 20

What are the TWO TYPES of GEL ELECTROPHORESIS?

Answer 20

1. Vertical Gel Electrophoresis
2. Horizontal Gel Electrophoresis

Question 21

the MOLECULAR SIZE of nucleic acid is expressed in MOLECULAR WEIGHT equivalent to the number of bases/ base pairs in the molecule.

Answer 21

Size of the Molecule

Question 22

A LINEAR DNA FRAGMENT of a given size migrates at different rates THROUGH GELS
containing different concentrations of agarose.

Answer 22

Agarose concentration

Question 23

The SUPERCOILED CIRCULAR DNA, relaxed circular DNA, and linear DNA of the same
molecular weight will migrate at different rates through the gel

Answer 23

DNA conformation

Question 24

The rate of migration is proportional to the VOLTAGE APPLIED ↑voltage ↑rate of migration.

Answer 24

Voltage applied

Question 25

It is the composition and IONIC STRENGTH AFFECTS DNA mobility.

Answer 25

Electrophoresis Buffer

Question 26

It dyes USED TO STAIN DNA in gels are usually intercalating agents.

Answer 26

Presence of DNA stains in the gel electrophoresis buffer

Question 27

Types of AGAROSE

Answer 27

* 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

Question 28

UV TRANSILLUMINATOR to view the fluorescent DNA in the gel.

Answer 28

Detection of DNA

Question 29

DYE AND SIZE must be the same as DNA being measured.

Answer 29

Quantification of DNA

Question 30

The bond appears as compact, with NO DOUBLE BOND or FAINT BAND.

Answer 30

Evaluation of the Quality of DNA

Question 30

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.

Answer 30

Genome Sequencing

Question 31

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.

Answer 31

DNA Shearing

Question 32

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.

Answer 32

DNA bar-coding

Question 33

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.

Answer 33

Whole genome sequencing

Question 34

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.

Answer 34

Data Analysis

Question 35

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

Answer 35

Gene Cloning

Question 36

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.

Answer 36

Southern-blot hybridization

Question 37

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.

Answer 37

Northern blot hybridization

Question 37

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.

Answer 37

Western Blot