topic 8: restriction endonucleases, RFLPS

Phosphodiester bonds

links nucleotides; make up the sugar phosphate backbone

Nucleotide

basic unit of nucleic acids

Nucleases

enzymes that break down nucleic acids via the hydrolysis of the phosphodiester bonds

Exonuclease

removes nucleotides from the free ends of the nucleic acid chains (ends only)

Endonuclease

hydrolyzes internal phosphodiester bonds; cut in between

Restriction Enzyme

An endonuclease, originally isolated from bacteria, that recognizes specific base sequences and cleaves the DNA polymer at the sugar-phosphate backbone.

BamHI

Bacillus amyloliquefaciens H

HindIII

Haemophilus influenzae Rd

SmaI

Serratia marcescense Sbb

​Methylation

The addition of a methyl group to a molecule; some restriction enzymes have this activity.

TRUE

T or F: binding and cleavage sites are not parts of the enzymes themselves but that of the nucleic acid

​Binding Site (Recognition Sequence)

The specific sequence of nucleotides identified and bound by a restriction endonuclease.

​Cleavage Site

The exact phosphodiester bond that is broken by a restriction enzyme.

GAATTC

EcoRI restriction enzyme

Type I Restriction Enzymes

both nuclease & methylase activity; bind to host specific DNA sites if 4-6 bp separated by 6-8 bp containing methylated adenine residues

Type III Restriction Enzymes

resembles Type I; have the ability to methylate & restrict (cut); adenine methylation only occurs in one strand; multiple subunits (helicase

unwinding activity); asymmetrical recognition sites; cleavage occurs 24-26 bp from the site to the 3’ site

Type IV Restriction Enzymes

have cutting & methyltransferase fxns

Type II

found in almost all prokaryotes; used most frequently in the laboratory; do not have inherent methylation activity; bind a simple dimer to symmetrical 4-8 bp DNA recognition sites; palindromic (bilateral symmetry); cleaves DNA directly at binding site; produce fragments of predictable size
Palindromic

​Sticky Ends

Single-strand overhangs (typically 2 to 4 bases) at the ends of DNA fragments produced by a staggered cut at the recognition site; can hybridize with the complementary ends of other DNA fragments; requires matching overhangs

DNA polymerase, Nucleotides as template, & ssExonuclease

used to convert sticky to blunt ends

​Blunt (Flush) Ends

DNA ends formed when a restriction enzyme cuts both strands of the duplex at the same position, leaving no overhangs.

​Adaptors

Short DNA fragments featuring one blunt end and one sticky end used to convert blunt ends into specific sticky ends.

Type IIS Restriction Endonucleases

recognizes 5-7 bp non-palindromic sequences; cuts DNA within 20 bases of the recognition site

Type IIT Enzymes

composed of two subunits, each containing one catalytic site

Type IIM Restriction Enzymes

Cut methylated DNA

​Restriction Fragment

A segment of DNA generated when a restriction enzyme cleaves a DNA strand.

Polymorphism

Variations in DNA sequences among individuals.

​RFLP (Restriction Fragment Length Polymorphism)

A technique using restriction enzymes to identify variations in homologous DNA sequences by observing differences in fragment sizes and numbers.

Southern Blot

reveals RFLPs by analysis of restriction fragments

Point Mutations

single base changes that can destroy EcoRI site

TGGCCA

recognition site of BalI

DNA

inherited as one haploid chromosome complement from each parent

Genetic Variations

increased due to mutations, intra- & inter- chromosomal recombination, gene conversion, & other genetic events

Single locus

have several versions or alleles

Homozygous

both alleles are the same

Heterozygous

two alleles are different

Mendelian Fashion

polymorphisms are inherited through this

Polymorphisms

can be used as landmarks or markers in the genome to determine the location of other genes

Formal statistical methods

used to determine the probability that an unknown gene is located close to a known marker in the genome

ABO blood group Ags

first genetic tool used for human ID

Tandem repeat

direct repeat of one to more than 100 nucleotides in length

GGCC

HaeIII

GANTC

HinfI

Southern Blot multiple-locus probe (MLP)-RFLP system

first human DNA profiling system introduced; utilizes 3-5 probes to analyze 3-5 loci on the same blot

Single locus probe

allows analysis of one locus at a time; easier to interpret

RFLP Southern Blot Technique

required 100 ng to 1 ug of relatively high-quality DNA; require fragile, large 0.7% gels; 32P bp system takes 5-7 days