Nucleic Acids and Proteins Flashcards by Keizel Galupo

Degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand.

nucleases

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Two kinds of nucleases

Exonuclease
Endonuclease

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Nucleases vary in

Specificity

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May be specific for DNA or RNA, such as DNases or RNases, respectively, or even be specific for a DNA/RNA hybrid, such as RNase H, which cleaves the RNA strand of a DNA-RNA hybrid. Therefore, its specificity varies dramatically.

nucleases

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Remove nucleotides one at a time from the end of a DNA molecule

Exonuclease

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May either attack a polynucleotide chain from the 5’ end and hydrolyze 5’ to 3’ or attack from the 3’ end and hydrolyze 3’ to 5’

Exonuclease

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When a nuclease hydrolyzes an ________________ bond in a ________________ linkage, it will have specificity for either of the two ester bonds, generating either 5’ nucleotides or 3’ nucleotides

ester; phosphodiester

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Nucleases may be specific for single strand nucleotide chain, double-helix stands, or both.

Strand preference

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An exonuclease that can remove nucleotides from both strands of a double-stranded molecule

Bal31

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An enzyme that degrades just one strand of a double-stranded molecule, leaving single-stranded DNA as the product

Exonuclease III

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Can hydrolyze internal bonds within a polynucleotide chain (break in the middle)

Endonuclease

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Can attack the phosphodiester bond from the 5’ end or from the 3’ end of the linkage

Endonuclease

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________________ endonuclease only cleaves single strands, whereas ________________ cuts both single- and doublestranded

S1; deoxyribonuclease I (DNase I)

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Recognize a specific nucleotide sequence and cleave the DNA molecules internally

Restriction endonucleases

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More commonly used in the lab

Restriction endonucleases

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3 classes of restriction endonucleases are distinguished by

mode of action

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Class of restriction endonuclease that are rather complex and have only a limited role in practical biotechnology applications.

Type I and Type III

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Class of restriction endonucleases which are the cutting enzymes that are so important in laboratory and clinical analysis.

Type II

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Type II restriction endonuclease is important because these sites (2) are the same.

recognition site
cleavage site

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Length of recognition/cutting sites

4 to 8 bp

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This means reading the same forward and backward on complementary strands

inverse palindromic

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Different sources of Type II restriction

Isoschizomers
Neoschizomers
Isocaudomers

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Q1: recognize and cut DNA at the same site

Q2: produce the same nucleotide extensions but have different recognition sites

Q3: recognize and bind to the same sequence of DNA but cleave at different positions

Isoschizomers;
Isocaudomers;
Neoschizomers

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Isoschizomers species

BspEI from a Bacillus species

AccIII from Acinetobacter calcoaceticus

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Neoschizomers species

**NarI** from Nocardia argentinensis **SfoI** from Serratia fonticola

Isocaudomers species

**NcoI** from Nocardia corallina **PagI** from Pseudomonas alcaligenes

represents a linear sequence of the sites at which particular restriction enzymes find their targets

Restriction map

Before sequencing a large stretch of DNA, this is done to **locate the cutting site** and **examine sizes of fragments**

Preliminary mapping

Q1: Restriction digest can be performed in a ________________ in the presence of all necessary components Q2: what are these components

microcentrifuge tube; template DNA, restriction enzyme, Mg2+

Q1: A restriction digest results in a number of ________________ Q2: the sizes of which depend on the

DNA fragments; positions of the recognition sequences

Used to analyze restriction digested DNA fragments

gel electrophoresis ***separated based on their size***

A specific **DNA** region can be analyzed by

Southern blot

Enzymes that **synthesize a new strand of DNA** complementary to an existing DNA or RNA template

Polymerases

Types of polymerases

1. DNA Polymerase I 2. Klenow Fragment 3. Taq DNA polymerase 4. Reverse Transcriptase

Prepared from **E. coli** which an example of an enzyme with a **dual activity**

DNA polymerase I

DNA polymerase I activities (2)

DNA polymerization DNA degradation

This treatment of DNA polymerase I produces two polypeptides

Mild proteolytic treatment

The **large fragment** produced by mild proteolytic treatment of DNA polymerase I which has the **polymerase 3' to 5' exonuclease activity**

Klenow fragment

A Klenow fragment can synthesize a complementary DNA strand on a single-stranded template also known as a ________________ region.

nick

Major application of Klenow fragment

Perform DNA end-filling or DNA sequencing

Bacterium of Taq DNA polymerase

Thermus aquaticus

Thermostable thus suitable for PCR

Taq DNA Polymerase

uses RNA as a template not DNA to synthesized a complementary DNA strand

Reverse transcriptase

newly synthesized DNA

complementary DNA (cDNA)

Q1: Used to evaluate **amount of RNA** Q2: Used to establish the **expression profile** (to evaluate the change of gene expression pattern)

Reverse transcriptase

modify DNA molecules by addition or removal of specific chemical groups

DNA modifying enzymes

Alkaline phosphatase; Polynucleotide kinase; Terminal deoxynucleotidyl transferaseadds

From **calf thymus disease** that adds one or more deoxyribonucleotides onto the 3' terminus of a DNA molecule

Terminal deoxynucleotidly transferaseadds

From **E coli. infected with T4 phage** that has reverse effect to alkaline phosphatase, **adds phosphate groups** onto free 5' terminus

Polynucleotide kinase

From **E. coli, calf intestinal tissue, or arctic shrimp** that **removes the phosphate group** present at the 5' terminus of a DNA molecule

Alkaline phosphatase

To simplify: **Alkaline phosphatase**: removes 5' phosphate group **Polynucleotide kinase**: adds or attaches 5' phosphate groups **Terminal deoxynucleotidyl transferaseadds**: attachees deoxyribonucleotides to the 3' termini

Repair single-stranded breaks (“discontinuities”) that arise in double-stranded DNA molecules during DNA replication or during DNA damage repair

DNa ligase

**Joins** two individual fragments of double-stranded DNA and individual DNA molecules

DNA ligase

Formation of hydrogen bonds between two complementary strands of nucleic acids

Hybridization

Q1: labeled strand Q2: process called

Probe; Hybridization (assay)

Formed between a labeled and unlabed strand

hybrid molecule

Reaction that is used to analyze the nucleic acid content of an unknown sample

hybridization assay

Hybridization assays

Southern Northern Dot/Blot Microarray Fluorescent in situ

Functions: 1) used to identify homologous sequences in **genomic DNA** 2) facilitate **gene mapping** through restriction mapping of genes 3) detection of restriction **fragment length polymorphisms**

Southern Hybridization

Southern Hybridization process

1. Extraction & Purification 2. Enzyme Digestion (product = DNA fragments) 3. DNA denaturation & fragmentation 4. Transfer / Blotting 5. Hybridization with labeled DNA probe 6. Detection & identification

Visualization of this means should show a series of smear bands without any discrete, distinguishable bands

visualized by staining

Parameters for Southern Hybridization

1) resolution of the agarose gel 2) depurination of DNA molecules 3) physical transfer of DNA onto the membrane support 4) fix DNA onto the **nylon membrane**

Southern Hybridization Q1: percentage of agarose gel Q2: voltage applied in gel electrophoresis

0.7 - 1.2%; High voltage, short runs: 10-20 kbp Low voltage, long runs: 1 and 10 kbp

Most critical step in Southern Hybridization

Depurination of DNA molecules

depurination by ________________________ upon denaturation by sodium hydroxide **leads to nicks** in the DNA strands, resulting in a **breakdown of long DNA fragments into shorter pieces** of single-stranded DNA.

0.2 M hydrochloric acid

Transfer methods

1) Classical Southern Transfer (ascending capillary transfer) 2) Descending Capillary transfer

Transfer of DNA to membrane in Southern Hybridization

Ascending capillary transfer

Uses the **gravitational flow of the transfer buffer with the help of vacuum** to allow more rapid and reproducible blotting of DNA

Descending capillary transfer

Fixing DNA onto the nylon membrane can be achieved by baking the membrane at or by

at 80C for 2 hrs or by UV light irradiation

Storage for baked or UV-fixed southern blot

room temp

Functions 1) allows detection of a given RNA molecules in a mixture of heterogeneous **RNA** 2) **Uses DNA probes to hybridize** with complementary RNA sequences 3) an ideal tool to study the **products of gene transcription**

Northern Hybridization

Northern Hybridization Before electrophoresis, denaturation is achieved by heating sample to

55C in the presence of **formaldehyde and formamide**

Its addition to the gel prevents reformation of secondary structure

formaldehyde

not usually recommended to add in electrophoresis, as it will always decrease the hybridization signal compared with unstained RNA

ethidium bromide

blotting for northern hybridization

capillary blotting or vacuum blotting

Rna can be fixed by

UV light irradiation or baking at 80C

Denaturation time for RNa Voltage and time

15 minutes 70V for 3.5 h

Results from a **variable number of tandem repeats** (VNTR) in a short DNA segment Used in **DNA fingerprinting and in paternity testing** Useful as a **genetic disease marker**

Restriction Fragment Length Polymorphism (RFLP)

performed when genomic DNA is **collected and is digested with a specific restriction enzyme** followed by gel electrophoresis

RFLP analysis

permits the simultaneous evaluation of **hundreds to thousands of different DNA regions**; useful in **nonmodel** species

Amplified Fragment Length Polymorphism (AFLP)

Allow high-resolution genotyping of fingerprinting quality

AFLP analysis

Factors that influence hybrid stability

Ionic Strength Base composition Destabilizing agents Mismatched base pairs Duplex length

Must be labed with a radioactive or other type of marker

probe

probes are denatured by

heating

applied to the membrane in a solution of chemicals that **promote nucleic acid hybridization**

Probes

An organic molecule that has a high affinity for a protein called avidin

biotin

a variation of the dot/slot blot in which the dotted material is arranged in a regular gridlike pattern

mircroarray

This technology is an analysis of nucleic acid on a genome-wide scale

microarray

Most comon application of microarray technology which is the gene-by-gene determination of differences

transcript profiling

Process of transferring/blotting the proteins from the gel onto the surface of an absorbent membrane to render the proteins accessible for the identifying ligand

Western Blot

proteins are fully denatured with SDS and heat in the presence of β-mercaptoethanol

SDS page

native proteins are separated in a gel containing a pH gradient works well for hydrophilic proteins

Isoelectric focusing (IFE)

requires no buffer tank and no cooling system

semidry blotting

most commonly used membrane for protein blotting;

nitrocellulose

has much superior mechanical strength than nitrocellulose; Its protein binding capacity is good and it can be obtained as positively charged membrane that even increases the binding and retention capacity

Nylon

Membrane of choice for many applications of Western blotting It combines high protein binding capacity with excellent mechanical resistance and good staining properties.

PVDF

Standard stains of similar sensitivity

Amino black and india ink

Stain with lowest sensitivity but convenient as it is reversible by immersing in 0.1 N NaOH

ponceau

gives optimal blocking with lowest background and without impairing immunoreactivity

Ovalbumin/gelatin

proved to be an economical and effective blocker

fat free milk

blocking is very simple but has a tendency to give elevated background; may mask or detach immunoreactive proteins

tween-20

method for western blot

indirect immunodetection

used in western blots which produce a colored precipitate

chromogen

commonly used enzymes for western blot

horseradish peroxidase (HRP) and alkaline phosphatase (AP)