[2S] UNIT 5 Characterization of Nucleic Acid Flashcards
Degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand.
Nucleases
NUCLEASES SPECIFICITY
Specific for DNA
DNAse
NUCLEASES SPECIFICITY
- Targets RNA
- Degrades all RNA
RNAse
NUCLEASES SPECIFICITY
- Able to cleave DNA and RNA hybrid
- Subtype of RNase
RNAse H
NUCLEASES SPECIFICITY
Degrades RNA bound covalently to DNA
RNAse H
When a nuclease hydrolyzes an ester bond in a phosphodiester linkage, it will have a specificity for either of the two ester bonds. This generates what nucleotides?
5’ nucleotides or 3’ nucleotides.
For single strand molecules (RNA), ______ can rapidly degrade RNA molecules into ribonucleotide subunits.
ribonuclease
TWO TYPES OF NUCLEASE
Hydrolyze internal bonds within a polynucleotide chain
Endonuclease
TWO TYPES OF NUCLEASE
● Cut the length of the DNA sequence
● Break internal phosphodiester bonds
Endonuclease
TWO TYPES OF NUCLEASE
Cut at any point depending on its target site
○ Usually in the middle portion of the fragment
○ Between the 5’ and 3’ terminus
Endonuclease
TWO TYPES OF NUCLEASE
Produce several segments of our polynucleotide
Endonuclease
TWO TYPES OF NUCLEASE
Target and remove the terminal nucleotide
Exonuclease
TWO TYPES OF NUCLEASE
Removes nucleotides one at a time from the end of a DNA molecule
Exonuclease
TWO TYPES OF NUCLEASE
Cuts the terminal nucleotide whether it is on the 5’ or 3’ (only at the end of the fragment)
Exonuclease
MECHANISM OF NUCLEASE HYDROLYSIS
Removes nucleotides from both strands of a double-stranded molecule
Bal31
MECHANISM OF NUCLEASE HYDROLYSIS
Progressive shortening of the dsDNA from both ends after treatment
Bal31
MECHANISM OF NUCLEASE HYDROLYSIS
● Removes nucleotides from the 3’ terminus
● Can only cut through double strands
Exonuclease III
MECHANISM OF NUCLEASE HYDROLYSIS
Cleaves only single-stranded DNA, including single-stranded nicks in mainly double-stranded molecules
S1 Nuclease
MECHANISM OF NUCLEASE HYDROLYSIS
Can only cut single strands nucleotides
○ Create a nick in our double stranded nucleotide
○ Cannot entirely cut the sequence (double
stranded)
S1 Nuclease
MECHANISM OF NUCLEASE HYDROLYSIS
Refers to a specific type of discontinuity
Nick
MECHANISM OF NUCLEASE HYDROLYSIS
You need another digestion for that exposed single strand to fully cut the segment
○ However, if your DNA is single stranded, it
can entirely create fragments
S1 Nuclease
MECHANISM OF NUCLEASE HYDROLYSIS
Cleaves both single and double-stranded DNA
DNAse I
MECHANISM OF NUCLEASE HYDROLYSIS
Depending on which part it attaches to or targets
○ Can cleave segments or several segments
DNAse I
MECHANISM OF NUCLEASE HYDROLYSIS
Produce mononucleotide
DNAse I
MECHANISM OF NUCLEASE HYDROLYSIS
Non-specific nuclease (can cut phosphodiester bond)
DNAse I
MECHANISM OF NUCLEASE HYDROLYSIS
Enables to characterize nucleic acid sequences if it contains the specific sequence
Restriction Endonuclease
MECHANISM OF NUCLEASE HYDROLYSIS
● Very much utilized
● Can recognize and cut specific nucleotide sequence
○ Can cleave DNA molecules internally
Restriction Endonuclease
ENDONUCLEASES FOR CUTTING DNA
It was shown that some strains of bacteria are immune to bacteriophage infection
○ Host defense mechanism
Restriction Endonuclease
ENDONUCLEASES FOR CUTTING DNA
T/F: Initial observation that led to the eventual discovery of restriction endonucleases was made in the early 1960s
F; 1950s
ENDONUCLEASES FOR CUTTING DNA
Restriction endonuclease are found only in?
Microorganisms
ENDONUCLEASES FOR CUTTING DNA
Occurs because bacterium produces an
enzyme that degrades the phage DNA before it has time to replicate and direct synthesis of new phage particles
Restriction
ENDONUCLEASES FOR CUTTING DNA
Degradative enzyme is called _______ _________ synthesized by many species of bacteria
restriction endonucleases
ENDONUCLEASES FOR CUTTING DNA
T/F: Bacterium’s DNA carries additional methyl groups that protect and prevent the degradative enzyme action
Restriction Endonuclease
ENDONUCLEASES FOR CUTTING DNA
T/F: Roughly round 2000 distinct restriction enzymes have been identified in the bacteria
Restriction Endonuclease
ENDONUCLEASES FOR CUTTING DNA
Function as homodimer; recognize symmetrical dsDNA (palindromes)
Restriction Endonuclease
ENDONUCLEASES FOR CUTTING DNA
Utilized in the digestion of DNA molecules for hybridization procedures or in the direct identification of mutations
Restriction Endonuclease
ENDONUCLEASES FOR CUTTING DNA
Recognize specific sequences of 4, 5, or 6 nucleotides
Restriction Endonuclease
ENDONUCLEASES FOR CUTTING DNA
Cut by breaking the phosphodiester bond in both strands
Restriction Endonuclease
ENDONUCLEASES FOR CUTTING DNA
T/F: Cutting genomic DNA with a RE results in many fragments of different sizes
T
ENDONUCLEASES FOR CUTTING DNA
The smaller the recognition sequence the larger the number of fragments produced
Restriction Endonuclease
ENDONUCLEASES FOR CUTTING DNA
T/F: RE recognizes palindromes
T
ENDONUCLEASES FOR CUTTING DNA
Reads the same in both directions
○ Sequences directly opposite one another on opposite strands of the dsDNA molecule
Palindromes
TYPES OF RESTRICTION ENDONUCLEASES
● Cleaves DNA at random sites far from its recognition sequence
● Non specific
Type I
TYPES OF RESTRICTION ENDONUCLEASES
Cleaves DNA at defined positions close to or within its recognition sequence
TYPES OF RESTRICTION ENDONUCLEASES
● Most employed: due to where it cleaves unlike types I and III which have different target sites from their recognition sequences
● Most used
● More specific
Type II
TYPES OF RESTRICTION ENDONUCLEASES
Cleaves outside its recognition sequence with both REase and MTase enzymatic activities in the same protein
Type IIG
TYPES OF RESTRICTION ENDONUCLEASES
Cleaves symmetric targets and cleavage sites
Type IIP
TYPES OF RESTRICTION ENDONUCLEASES
Recognizes asymmetric sequences
Type IIS
TYPES OF RESTRICTION ENDONUCLEASES
Cleaves outside its recognition sequence and require two sequences in opposite orientations within the same DNA
Type III
TYPES OF RESTRICTION ENDONUCLEASES
Cleaves modified (e.g. methylated) DNA
Type IV
T/F: RE can’t protect bacterial cell from phage infection
F; can protect
RESTRICTION ENDONUCLEASE (Type II)
Can recognize the palindromic GAATTC sequence (hexanucleotide)
○ Needs to be palindromic to the complementary or opposite strand
E.COLI RESTRICTION ENZYME I (ECORI)
RESTRICTION ENDONUCLEASE (Type II)
Recognition Site of ECORI
Sequence
RESTRICTION ENDONUCLEASE (Type II)
Cleavage Site of ECORI
between the G and A (both sense and antisense strand)
RESTRICTION ENDONUCLEASE (Type II)
Produces staggered or cohesive cut (sticky ends)
ECORI & PSTI
RESTRICTION ENDONUCLEASE (Type II)
Overhang of ECORI
○ Left: Overhang at the bottom
○ Right: Overhang at the top
5’ Overhang
RESTRICTION ENDONUCLEASE (Type II)
Can recognize the palindromic CTGCAG sequence
PSTI
RESTRICTION ENDONUCLEASE (Type II)
Obtained from Providencia stuartii
PSTI
RESTRICTION ENDONUCLEASE (Type II)
Cleavage Site of PSTI
between A and G (and the other
strand)
RESTRICTION ENDONUCLEASE (Type II)
Overhang of PSTI
3’ Overhang
RESTRICTION ENDONUCLEASE (Type II)
Obtained from Arthrobacter luteus
ALUL
RESTRICTION ENDONUCLEASE (Type II)
Can recognize the AGCT sequence (four palindromic nucleotide)
ALUL
RESTRICTION ENDONUCLEASE (Type II)
Cleavage Site of ALUL
in between C and G (as well as for the
other strand)
RESTRICTION ENDONUCLEASE (Type II)
Produces a blunt cut
ALUL
RESTRICTION ENDONUCLEASE (Type II)
No cohesive ends and overhang are produced
ALUL
RESTRICTION ENZYMES
● Uneven cleavage
● 5’Overhaul
BamH1
RESTRICTION ENZYMES
● Recognizes GGATCC
● Cutting in between two G’s
○ Cohesive or Sticky ends
BamH1
RESTRICTION ENZYMES
DpnI, HaeIII
METHYLATION-SENSITIVE ENZYMES
RESTRICTION ENZYMES
BamHI, BG1II
ENZYMES GENERATING COMPATIBLE COHESIVE ENDS
RESTRICTION ENZYMES
● Recognizes GGTACC
● Cutting in between two C’s
● 3’ Overhang
Kpnl
RESTRICTION ENZYMES
● Recognizes GGCC
● Blunt Ends
● No Overhang
HaeIII
RESTRICTION ENZYMES
produces staggered cut
BamHI, BG1II
RESTRICTION ENZYMES
■ BamHI: GGATCC
■ BglII: AGATCT
ENZYMES GENERATING COMPATIBLE COHESIVE ENDS
RESTRICTION ENZYMES
Produce compatible overhangs upon cutting
ENZYMES GENERATING COMPATIBLE COHESIVE ENDS
RESTRICTION ENZYMES
Produced from Haemophilus spp.
RESTRICTION ENZYMES
T/F: If a sequence is methylated, it is unable to cut
T
RESTRICTION ENZYMES
○ ____: requires methylation to function
○ ____: inhibited by methylation
DpnI
HaeIII
RESTRICTION ENZYMES
principle of bacteria preserving its own
DNA
Methylation
RESTRICTION ENZYMES
When these nucleases were produced, this may be a response to the ___________
○ (Ex.) GATC (A is methylated)
methylation
RESTRICTION ENZYMES
T/F: All of the restriction endonucleases requires methylated nucleotides
F; some not all
DIFFERENT SOURCES OF TYPE II RESTRICTION ENDONUCLEASES
Recognize and cut DNA at the same site or palindromic sequence but cuts differently
Isoschizomers
DIFFERENT SOURCES OF TYPE II RESTRICTION ENDONUCLEASES
○ BspEI from a Bacillus species
○ AccIII: Acinetobacter calcoaceticus
Isoschizomers
DIFFERENT SOURCES OF TYPE II RESTRICTION ENDONUCLEASES
SmaI, XmaI
○ (Ex.) CCC|GGG producing blunt ends ( | as blunt end) in Smal
○ (Ex.) XmaI targeting the same recognition site
Isoschizomers
DIFFERENT SOURCES OF TYPE II RESTRICTION ENDONUCLEASES
Recognize and bind to the same sequence of DNA but cleave at different position
Neoschizomers
DIFFERENT SOURCES OF TYPE II RESTRICTION ENDONUCLEASES
○ NarI: Nocardia argentinensis
○ SfoI: Serratia fonticola
Neoschizomers
DIFFERENT SOURCES OF TYPE II RESTRICTION ENDONUCLEASES
Different single stranded extensions
Neoschizomers
DIFFERENT SOURCES OF TYPE II RESTRICTION ENDONUCLEASES
Restriction endonucleases that have the same nucleotide extensions (or overhangs) but have different recognition sites
Isocaudomers
DIFFERENT SOURCES OF TYPE II RESTRICTION ENDONUCLEASES
○ NcoI: Nocardia corallina
○ PagI: Pseudomonas alcaligenes
Isocaudomers
Governs the sequence of recognition
Restriction-Modification System / (R-M) System
Each endonuclease has its own pair of methylase
Restriction-Modification System / (R-M) System
○ Protective mechanism of bacteria for preventing its own DNA from being cut or degraded by its own restriction enzymes
○ Important during replication
Restriction-Modification System / (R-M) System
RESTRICTION-MODIFICATION SYSTEM
Which strand is methylated?
Parent Strand
RESTRICTION-MODIFICATION SYSTEM
T/F: Almost all restriction endonucleases are paired with methylases that recognize and methylate the same DNA sites
T
THE FREQUENCY OF RECOGNITION SEQUENCES IN A DNA MOLECULE
Can be performed in a microcentrifuge tube
in the presence of all necessary components including:
■ Template DNA
■ Restriction enzyme
■ Mg2+ at the right conditions
Restriction Digest
Both restriction endonucleases and methylases are collectively called a _________
Restriction-Modification System / (R-M) System
ANALYSIS OF RESTRICTION DIGESTED FRAGMENTS
T/F: RD results in a number of DNA fragments. Sizes depend on the exact positions of the recognition sequences for the endonuclease in the original molecule and can be analyzed by gel electrophoresis
T
Enzymes synthesizing a new strand of DNA complementary to an existing DNA or RNA template
Polymerases
Most function only if the template possesses a double-stranded region that acts as a primer for initiation of polymerization.
Polymerases
POLYMERASES
Prepared from E. coli
DNA Pol I
POLYMERASES
DNA polymerase activity: attach to a short
single-stranded region (or nick) in a mainly double stranded DNA molecule (synthesizes a completely new strand)
DNA Pol I
POLYMERASES
Exonuclease activity
○ 3’-5’: proofreading newly synthesized DNA
○ 5’-3’: degrade a strand / remove and replace strand
DNA Pol I
POLYMERASES
DNA polymerization and DNA degradation
DNA Pol I
POLYMERASES
● Large fragment
○ 3’- 5’ exonuclease activity and no 5’-3’ exonuclease activity
● Small fragment
○ 5’-3’ exonuclease activity
Klenow Fragment
POLYMERASES
Synthesize a complementary DNA strand
(nick region)
Klenow Fragment
POLYMERASES
DNA end-filling or DNA sequencing
Klenow Fragment
POLYMERASES
Used in the PCR as the DNA polymerase I enzyme of the bacterium Thermus aquaticus
Taq DNA Pol
POLYMERASES
Thermostable: resistant to denaturation by heat
○ Suitable for PCR: -94° C (denature the DNA)
○ Resistant to denaturation by heat treatment
Taq DNA Pol
POLYMERASES
Would not be inactivated when the temperature of the reaction was raised to 94° C to denature the DNA
Taq DNA Pol
POLYMERASES
● Replication of virus
○ Uses RNA as a template not DNA
○ Synthesize complementary DNA (cDNA)
Reverse Transcriptase
POLYMERASES
● Evaluate the amount of RNA
● Establish the expression profile
● Change in gene expression pattern
Reverse Transcriptase
NUCLEIC ACID MODIFYING ENZYMES
Numerous enzymes modify DNA molecules by addition or removal of specific chemical groups
DNA Modifying Enzymes
NUCLEIC ACID MODIFYING ENZYMES
Repair single-stranded breaks (discontinuities)
○ Arise in double-stranded DNA molecules
during DNA replication or during DNA damage repair
DNA Ligase
NUCLEIC ACID MODIFYING ENZYMES
Can also join together two individual fragments of double-stranded DNA
DNA Ligase
NUCLEIC ACID MODIFYING ENZYMES
Catalyses formation of bonds between 5’-P and 3’-OH groups on backbone of DNA
DNA Ligase
NUCLEIC ACID MODIFYING ENZYMES
● Ligate “blunt end” or “sticky ends”
● Repair “nicks” in DNA
DNA Ligase
NUCLEIC ACID MODIFYING ENZYMES
Require primers to extend and copy DNA
DNA Polymerase
NUCLEIC ACID MODIFYING ENZYMES
● All extend 5’→3’ by adding on to 3’-OH
● Make a reverse, complimentary copy
DNA Polymerase
NUCLEIC ACID MODIFYING ENZYMES
T/F: Two Reactions Catalyzed by DNA Ligase
1) DNA ligase repair of discontinuity. A missing phosphodiester bond in one strand of a double-stranded molecule
2) DNA ligase joining molecules
T
NUCLEIC ACID MODIFYING ENZYMES
Cut DNA in non-sequence specific manner
Nucleases - Exonucleases
NUCLEIC ACID MODIFYING ENZYMES
Can digest DNA from either 5’-3’ or 3’-5’ direction
Nucleases - Exonucleases
NUCLEIC ACID MODIFYING ENZYMES
● Prefer ssDNA
● Proofreading function of polymerase Alkaline phosphatase
Nucleases - Exonucleases
NUCLEIC ACID MODIFYING ENZYMES
Removes 5’ P: prevents recircularization of plasmids
Alkaline Phosphate
NUCLEIC ACID MODIFYING ENZYMES
Phosphatases (dephosphorylate 5’-terminus of DNA molecule)
Alkaline Phosphate
NUCLEIC ACID MODIFYING ENZYMES
● Digest DNA molecules: non-specifically digests dsDNA or ss DNA
● Commonly found on most surfaces, including hands
DNAse
NUCLEIC ACID MODIFYING ENZYMES
digest RNA molecule
RNAse: Ribonuclease
NUCLEIC ACID MODIFYING ENZYMES
● Many different types, may be specific for ssRNA or RNA/DNA hybrids (RNAse H)
● Extremely common (especially on hands), very stable
RNAse
THE APPLICATION OF NUCLEIC ACID HYBRIDIZATION
Formation of hydrogen bonds between
two complementary strands of nucleic acids
Hybridization
THE APPLICATION OF NUCLEIC ACID HYBRIDIZATION
T/F: Binding between separate, complementary nucleic acids is both irreversible and base sequence-specific
F; reversible
THE APPLICATION OF NUCLEIC ACID HYBRIDIZATION
Direct consequence of the stable
double-stranded structure of nucleic acid
under physiological conditions
Hybridization
THE APPLICATION OF NUCLEIC ACID HYBRIDIZATION
T/F: During the annealing process, both nucleic acid strands are not labeled with any isotopes or fluorescence
T
THE APPLICATION OF NUCLEIC ACID HYBRIDIZATION
● Probe: labeled strand
● Process of labeling: hybridization
○ Hybrid molecule: formed between a labeled and unlabeled strand
● Hybridization assay: used to analyze the nucleic acid content of an unknown sample
Hybridization
TYPES OF NUCLEIC ACID HYBRIDIZATION
Detection of a given DNA sequence in a complex mixture of DNA sequences
Southern Hybridization
TYPES OF NUCLEIC ACID HYBRIDIZATION
Identify homologous sequences in genomic DNA
Southern Hybridization
TYPES OF NUCLEIC ACID HYBRIDIZATION
Facilitate gene mapping through restriction mapping of genes
Southern Hybridization
TYPES OF NUCLEIC ACID HYBRIDIZATION
Detect of restriction fragment length polymorphisms
Southern Hybridization
TYPES OF NUCLEIC ACID HYBRIDIZATION
Northern Blot Denaturing, Staining & Electrophoretic
Northern Hybridization
TYPES OF NUCLEIC ACID HYBRIDIZATION
Technique used to study gene expression by detecting specific RNA sequences
Northern Hybridization
TYPES OF NUCLEIC ACID HYBRIDIZATION
The availability of a variety of restriction endonuclease enzymes that cleave DNA at specific sites has made it possible to identify the presence of polymorphic regions in the isolated fragments
Restriction Fragment Length Polymorphism (RFLP)
TYPES OF NUCLEIC ACID HYBRIDIZATION
● In DNA fingerprinting
● In paternity testing
Restriction Fragment Length Polymorphism (RFLP)
TYPES OF NUCLEIC ACID HYBRIDIZATION
T/F: RFLP as a molecular marker is specific to a single clone / restriction enzyme combination
T
TYPES OF NUCLEIC ACID HYBRIDIZATION
Results from a variable number of tandem repeats (VNTR) in a short DNA segment
Restriction Fragment Length Polymorphism (RFLP)
TYPES OF NUCLEIC ACID HYBRIDIZATION
- Genomic DNA collected
- Digested with a specific restriction enzyme
- Gel electrophoresis
- Southern blot analysis
RFLP Analysis
TYPES OF NUCLEIC ACID HYBRIDIZATION
● Genetic disease marker
● Closeness to the disease gene
● Sufficient DNA
Restriction Fragment Length Polymorphism (RFLP)
TYPES OF NUCLEIC ACID HYBRIDIZATION
Cleaved amplified polymorphic sequence (CAPS) assay
Restriction Fragment Length Polymorphism (RFLP)
TYPES OF NUCLEIC ACID HYBRIDIZATION
● Fingerprinting technique
● Permits the simultaneous evaluation of different DNA regions distributed randomly throughout the genome without prior sequence knowledge
Amplified Fragment Length Polymorphism (AFLP)
TYPES OF NUCLEIC ACID HYBRIDIZATION
Useful in non-model species which have no complete genome sequences available and where other types of genome-wide markers are difficult to obtain
Amplified Fragment Length Polymorphism (AFLP)
DETECTION METHODS
Labeled, denatured single-strand
○ Label: radioactive or other type of marker
○ Denatured by heating
○ Applied to the membrane
Probe
DETECTION METHODS
● Hybridizes to membrane-bound DNA or RNA
● Promote nucleic acid hybridization
Probe
TYPES OF NUCLEIC ACID HYBRIDIZATION
Is highly reproducible and robust because it combines the specificity of RFLP with the sensitivity of the PCR
Amplified Fragment Length Polymorphism (AFLP)
DETECTION METHODS
Shows unique blotting pattern characteristic of a specific genotype at a specific locus
RFLP Probe
DETECTION METHODS
● Labeled DNA sequence
● Hybridizes with fragments of the digested DNA sample after gel electrophoresis
RFLP Probe
DETECTION METHODS
Typically short, single- or low-copy genomic DNA or cDNA clones
RFLP Probe
DETECTION METHODS
Used in genome mapping and variation analysis
RFLP Probe
DETECTION METHODS
DNA molecule is usually labeled by incorporating nucleotides that carry a radioactive isotope of phosphorus, 32P
Labeling With a Radioactive Marker
DETECTION METHODS
This reaction requires a supply of nucleotides, one of which is radioactively labeled with 32P-modified deoxynucleoside triphosphate
Labeling With a Radioactive Marker
4 Detection Methods
- Probe
- RFLP Probe
- Labeling With a Radioactive Marker
- Labeling With a Nonradioactive Marker
DETECTION METHODS
During the synthesis, the DNA molecule will become labeled as the radiolabeled deoxynucleotides are attached to the newly synthesized strand
Labeling With a Radioactive Marker
DETECTION METHODS
Probe DNA is complexed with the enzyme horseradish peroxidase
Nonradioactive hybridization probing
DETECTION METHODS
Deoxyuridine triphosphate (dUTP) nucleotides modified by reaction with biotin
Labeling With a Nonradioactive Marker
DETECTION METHODS: Labeling With a Nonradioactive Marker
An organic molecule that has a high affinity for a protein called avidin
Biotin
Reverse dot/slot blot of several thousand targets on nitrocellulose or nylon membranes
Macroarrays
Sample does not pass through gel electrophoresis, samples are instead directly applied onto your membrane: Arrangements include:
● Dot Blot and Slot Blot Hybridization
● Macroarrays
● Microarrays
● Microarray-Manufacturing Technology
● Sample Processing and Detection
Array-Based Hybridization
● Visualized without magnification
● Typically use radioactive or chemiluminescent signals
Macroarrays
Probes deposited onto the membrane by printing or dot blotting, then dried and stored for future use
Macroarrays
Limitations:
- area of the membrane
- specimen requirement
Macroarrays
Variation of the dot/slot blot iarranged in a regular gridlike pattern
Microarrays
● Microarray - delivery
● GeneChips - synthesis
Microarrays
The most common application of ______ technology is transcript profiling
Microarrays
Most critical in sample processing & detection
Isolation of mRNA from cells or tissues
Sample Processing and Detection
Surface plasma resonance
Unlabeled Probes
