Nucleic Acids and Proteins Flashcards
Degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand.
nucleases
Two kinds of nucleases
Exonuclease
Endonuclease
Nucleases vary in
Specificity
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
Remove nucleotides one at a time from the end of a DNA molecule
Exonuclease
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
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
Nucleases may be specific for single strand nucleotide chain, double-helix stands, or both.
Strand preference
An exonuclease that can remove nucleotides from both strands of a double-stranded molecule
Bal31
An enzyme that degrades just one strand of a double-stranded molecule, leaving single-stranded DNA as the product
Exonuclease III
Can hydrolyze internal bonds within a polynucleotide chain (break in the middle)
Endonuclease
Can attack the phosphodiester bond from the 5’ end or from the 3’ end of the linkage
Endonuclease
________________ endonuclease only cleaves single strands, whereas ________________ cuts both single- and doublestranded
S1; deoxyribonuclease I (DNase I)
Recognize a specific nucleotide sequence and cleave the DNA molecules internally
Restriction endonucleases
More commonly used in the lab
Restriction endonucleases
3 classes of restriction endonucleases are distinguished by
mode of action
Class of restriction endonuclease that are rather complex and have only a limited role in practical biotechnology applications.
Type I and Type III
Class of restriction endonucleases which are the cutting enzymes that are so important in laboratory and clinical analysis.
Type II
Type II restriction endonuclease is important because these sites (2) are the same.
recognition site
cleavage site
Length of recognition/cutting sites
4 to 8 bp
This means reading the same forward and backward on complementary strands
inverse palindromic
Different sources of Type II restriction
Isoschizomers
Neoschizomers
Isocaudomers
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
Isoschizomers species
BspEI from a Bacillus species
AccIII from Acinetobacter calcoaceticus
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
- DNA Polymerase I
- Klenow Fragment
- Taq DNA polymerase
- 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
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
- Extraction & Purification
- Enzyme Digestion (product = DNA fragments)
- DNA denaturation & fragmentation
- Transfer / Blotting
- Hybridization with labeled DNA probe
- 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)
