DLA Recombinant DNA Technology, Restriction Endonucleases And Vectors Flashcards

Question

Give a historical perspective of bacteriophages

Answer 1

Bacteriophage - Early 1960s, the ability of bacteriophage to incorporate their genes into host bacterial genome identified - Scientists noticed linear phage genomic DNA seem to form a circle upon entering host bacteria, break apart-rejoin within the host chromosome “recombination”

Answer 2

Reversal recombination and phage DNA is excised - found the phage DNA contained small complementary regions at the ends of single stranded DNA(ssDNA) - These regions called cohesive (cos) sites allow for complementary pairing of the DNA ends so that linear DNA can form a circle within the host - Joining bits of DNA by cohesive sites is the cornerstone of genetic engineering

Answer 3

Some phages which happily grow in one bacterial host did not grow in different strains, their GROWTH was RESTRICTED Phage grown in E. coli strain C has restricted growth when it infected a rare strain of E. Coli called strain K-12 - lambda dna degraded by K-12 - K-12 is the most well studied strain of bacteria (standard for modern molecular work) - Now phage called gamma-C phage as it was grown in E. Coli C - Occasionally, rare progeny of phage could escape “restriction “ where phage DNA is methylated - These phage became modified and would normally grow in K-12 cells called, phage gamma-k Phage survives and causes cell lysis

Answer 4

There are 2 enzymes that recognize particular sequence of DNA -One is methylase, an enzyme that methylates DNA at a particular sequence of DNA An endonuclease which recognizes the same sequence of DNA and will cleave the DNA but only if the DNA is NOT methylated - EcoRI is a restriction enzyme that cleaves DNA at the site GAATTC - bacterial DNA is modified by a methylase immediately after replication - Methylase is part of the bacterial restriction modification system - Methylase is an enzyme that recognizes the same target site as Restriction enzyme and adds a methyl group to a nucleotide in the restriction site - Offers protection from cleavage This allows protection in the E. Coli-k-12 strain

Answer 5

Bacteria methylate their own DNA in a series specific manner - Arber et al. demonstrated that this methylation protects DNA from degradation, so it is no longer recognized by restriction enzymes - When bacterial DNA replicates, old strand remains methylated and new strand is unmethylated - New strand is quickly methylated by specific methylase AND IS PROTECTED - gamma-k has modified DNA - Phage DNA NOT recognized as foreign (SAME methylation pattern) - Phage DNA replicates and is methylated by a specific methylase and pattern is maintained - DNA is not cleaved and gamma-K progeny are produced

Answer 6

- 1968, Meselson and yuan purified an enzyme that cleaved gamma-C DNA, 5 reproducible pieces, but not gamma-k DNA - this enzyme is called restriction endonuclease or restriction enzyme because it restricted the viral infection - Also called a nuclease(an enzyme that cleaves phosphodiester bonds within a nucleotide chain) in the bacteria had the ability to distinguish resident from foreign DNA - Phage DNA that enters the host cell either unmyelinated or had an alternate pattern of methylation from host is considered Foreign Foreign DNA is degraded by host restriction enzymes - gamma-C infects K-12 cell( which is resistant to phage) - Phage DNA recognized as foreign (different methylation pattern from host) - Phage DNA cleaved by a specific restriction enzyme - No progeny are produced

Answer 7

The basic tool of cloning - 3 major classes: grouping is based on type of sequence they recognize - Type 1 and type 3 not useful for cloning because they are promiscuous which causes random cleavage patterns - Type 2 have specific sites and cleave only at their site reproducibly

Answer 8

Less common that type 2 Recognition site: cuts both strands, nonspecific location, far from recognition site Composition: 3-subunit complex separate recognition, endonuclease & methylase Not useful in recombinant research

Answer 9

The most common in abundance Cuts both strands, specific, sites usually palindromic, 4-8 bp long - Endonuclease & methylation separate - Very useful

Answer 10

Rare in abundance Cuts one strand, 24-25 bp downstream of recognition site Endonuclease & methylase are separate, two-subunit complexes with one common subunit Not useful in recombinant research

Answer 11

Named for the organism where found using the 3 letters - 4th is the strain - Followed by Roman numerals for the order of discovery in that organism HindIII( his dee three) -discovered in haemophilus influenza, strain D and the third found EcoRI(echo r one) -from E. Coli, strain R and the first one found BamHI (Bam h one) -from bacillus amuloiquefaciens, strain H and the first one found

Answer 12

- Homodimers that recognize short, symmetric DNA sequences - 4-8 bp in length, 6 bp most common - Sequence read 5’—> 3’ on one side and 5’ —> 3’ on complimentary strand - Sequences read the same in both directions called palindromes (Greek for “run back”)

Answer 13

HindIII- “sticky end” with 5’ overhang Smal - “blunt end” with NO overhang EcoRI - “sticky end” with 5’ overhang BamHl- “sticky end” with 5’ overhang

Answer 14

Level Madam Rotator Deleveled

Answer 15

Restriction endonucleases possess recognition sequences that vary considerably in length. This property allows researchers to digest DNA into predictable sized units. -e.g. DNA with 50% G/C content and random sequence (hypothetical) Hpall recognized the sequence 5’ -CCGG- 3’ - There is a probability of 1/ 4(A,C,G, or T) occurring at a particular location - Therefore, the Hpall recognition site will occur at a frequency of (0.25)^4= 256 So, you expect that a Hpall recognition site of 4 bp will occur every 256 base pairs 6 bp recognition site= (0.25)^ 6= 1/4,096 8 bp recognition site= (0.25)^8= 1/65,536

Answer 16

- DNA joining enzyme called DNA ligase - Catalyses the formation of phosphodiester bond - 5’ phosohate of one nucleotide to the 3’ hydroxyl of the next nucleotide Restriction enzyme recognizes site Cleaves phosphodiester bonds BUT stocky ends (also called cohesive ends) can re-establish H-bonds DNA ligase re-forms the phosphodiester bonds (on both sticky ends and blunt ends)

Answer 17

- DNA to be cloned is cut with the same restriction enzyme - Plasmid vector is removed from bacterial cell and cut with a restriction enzyme —The two DNAs are ligates to form a recombinant molecule - Introduction into bacterial host cells by transformation Cells carrying recombinant plasmids can be selected by plating on medium containing antibiotics and color indicators such as X-gal DNA ligase uses ATP to form phosphodiester bonds

Answer 18

-Blunt end cutting has low efficiency of ligation in cloning, so we can add complementary sticky ends with the enzyme terminal deoxynucleotidyl transferase Add T-tail over hang and complementary A-tail overhang on other strand, making two sticky ends -Problem - restriction site is destroyed

Answer 19

Endonucleases- internal cuts Exonuclease- nucleotides removed from the ends

Answer 20

- use restriction mapping to characterize a particular piece of DNA - Usually first step in characterizing a piece of DNA - Double digests are more useful to characterize DNA

Answer 21

First stage analysis: after identifying a clone of interest to create a restriction map - Restriction map complies the number, order & distance between restriction sites of the cloned DNA fragment - restriction map is also important for characterizing DNA, for mapping genes and used for diagnostic tests for many genetic diseases

Answer 22

2 kB fragment in a vector and orientation of insert unknown 2 possibilities - Digest will tell the orientation - One EcoRl site, plasmid & insert is 5.8 kb - Cut with BamHl, vector is 3.8 kB and insert is 2kb - Cut with Hindlll and EcoRI and we know the orientation of the clone is the first example - If it was second orientation, we would have seen fragments of 3.5 and 2.3 kB

Answer 23

Homodimer Sliding/jumping until coupling occurs, Catalysis leads to enzyme being released and DNA cut

Answer 24

- first stage analysis: after identifying a clone of interest to create a restriction map - Restriction map complies the number, order & distance between restriction sites of the cloned DNA fragment - Restriction map also important for characterizing DNA, for mapping genes and 7sed for diagnostic tests for many genetic diseases