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Molecular Biology > DLA Recombinant DNA Technology, Restriction Endonucleases And Vectors > Flashcards

DLA Recombinant DNA Technology, Restriction Endonucleases And Vectors Flashcards

1
Q

What is biotechnology?

A

The manipulation of living organisms or their genetic components to produce goods and services

The major breakthrough is recombinant DNA technology that now enables foreign genes to be expressed in other organisms

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2
Q

What is recombinant DNA technology?

A

Nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule

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3
Q

What is a recombinant organism?

A

Possesses a different combination of alleles from its parents

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4
Q

What is a vector?

A

A piece of DNA that is capable of independent growth(often circular bacterial plasmids or viral phages)

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5
Q

What are the applications of red biotechnology?

A

Medical/pharmaceutical: red biotechnology

Agricultural applications: green biotechnology

Marine and aquatic applications: blue biotechnology

Industrial applications: white biotechnology

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6
Q

What is genetic engineering?

A

The direct manipulation of genes for practical purposes.

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7
Q

What is a clone?

A

A group of cells that contain the same recombinant DNA molecule

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8
Q

What is gene cloning?

A

Using bacteria to make multiple copies of a gene

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9
Q

Explain step by step gene engineering

A
  1. Gene inserted into plasmid
  2. Plasmid put into cell
  3. Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest
  4. Protein expressed from gene of interest. Basic research and various applications
    Such as:
    -gene for pest resustance inserted into plants
  • gene used to alter bacteria for cleaning up toxic waste
  • Protein dissolves blood clots in heart attack therapy
  • Human growth hormone treats stunted growth
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10
Q

What is agricultural biotechnology?

A

Agricultural scientists have endowed a number of crop plants and animals with genes for desirable traits thereby making genetically modified (GM) plants and animals, respectively

-Most public concern about possible hazards centers on genetically modified organisms (GMO) used as food

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11
Q

What are transgenic animals?

A

Animal biotechnology-Transgenic animals are made by introducing genes from one species into the genome of another animal

Animals are engineered to have desired characteristics

-Improve animals or their products

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12
Q

What are transgenic plants?

A

Genetic engineering in plants has been used to transfer many useful genes into plants

  • Plants are engineered to have desired characteristics
  • Improve plants and the products produced from them
  • The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
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13
Q

What is environmental biotechnology?

A
  • Any biotechnology process that may promote a good environment
  • Genetic engineering can be used to modify the metabolism of microorganisms

Some modified microorganisms can be used to:

  • Extract minerals from the environment(bio mining) or
  • Degrade potentially toxic waste materials. (Bioremediation)
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14
Q

Describe oil eating microbes

A

Naturally occurring microbes in the ocean feed on the hydrocarbons in oil. Scientists hope to speed up the process for the large spill on the Gulf of Mexico, where warm temperatures also aid in reaction

Oxygen is needed for the chemical reaction, but can be sparse at great ocean depths

The microbes break apart the hydrocarbons and combine them with oxygen to create water and carbon dioxide
-Not all of the oil is consumed,

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15
Q

What is white biotechnology?

A

Or industrial technology involves the use of cells (e.g. microorganisms) or components of cells (e.g. enzymes ) to process and generate industrially-useful products

Microorganisms like yeasts, mounds and bacteria can be improved (by genetic engineering) to work better as cell factories that efficiently produce industrial enzymes

 -E.g. genetically improved yeast or bacteria and enzymes are used to convert plant material into sugars that are in turn fermented to produce bio-ethanol
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16
Q

What is red biotechnology?

A

Using genetic engineering in medical processes to improve human health

Applications of red(medical) biotechnology:

  • drug production
  • pharmacogenomics
  • gene therapy
  • genetic testing (or genetic screening)
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17
Q

What is the sacrifice to replace human insulin ?

A

It takes 14 cows a year, to treat one diabetic a year

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18
Q

What is the sacrifice of producing human growth hormone?

A

It takes 80 human cadavers to treat one person/year with the risk of transmitting human pathogens

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19
Q

What is the sacrifice vitamin b2 in red biotechnology?

A

Produced traditionally from glucose in a multiple step chemical process

Currently produced enzymatically using yeast as a by-product of fermentation

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20
Q

How do scientists brew tetrahydrocannabinol (THC)?

A

They use yeast

Synthetic THC in pill form sold under brand names such as Cesamet.

-It’s often used to treat nausea associated with HIV or cancer therapies, but this new process could offer a less expensive, easier way to create this cannabis component from scratch

21
Q

How is vaccine production used for red biotechnology?

A

Making a polyvalent vaccine in Vaccinia

22
Q

What is gene therapy?

A

Gene therapy can be used to cure genetic diseases by replacing the defective or missing gene with therapeutic genes

Recombinant DNA techniques can be used to increase understanding of DNA for gene therapy

In vivo gene therapy

Ex vivo gene therapy

23
Q

What are restriction enzymes (endonucleases)?

A

An enzyme that cleaves (cuts) DNA at or near specific recognition nucleotide sequences known as restriction sites

24
Q

What are expression vectors(expression construct) ?

A

A plasmid or virus designed for protein expression occurs cells
-the vector is used to introduce a specific gene into a target cell, and can commandeer the cell’s mechanism for protein synthesis to produce the protein encoded by the gene

-The plasmid is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector
25
Q

Give a historical perspective of bacteriophages

A

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”
26
Q

How has the bacteriophage historically contributed to gene engineering?

A

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
27
Q

What are the bacterial restriction & modification systems?

A

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

28
Q

Explain enzyme function for bacterial restriction modification system

A

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

29
Q

How does the bacteria modify its own DNA?

A

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
30
Q

What was the contribution of Meselson and Yuan to bacterial modification and bacterial restriction?

A
  • 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
31
Q

What are restriction endonucleases?

A

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
32
Q

What are the characteristics of restriction endonuclease type 1?

A

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

33
Q

What are the character of type 2 restriction endonucleases ?

A

The most common in abundance

Cuts both strands, specific, sites usually palindromic, 4-8 bp long

  • Endonuclease & methylation separate
  • Very useful
34
Q

What are the characteristics of restriction endonucleases type 3?

A

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

35
Q

Explain the nomenclature of restriction endonucleases

A

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

36
Q

Explain the recognition sequences are recognized for type 2 restriction endonuclease

A
  • 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”)
37
Q

What are the recognition sequences for type 2?

A

HindIII- “sticky end” with 5’ overhang

Smal - “blunt end” with NO overhang

EcoRI - “sticky end” with 5’ overhang

BamHl- “sticky end” with 5’ overhang

38
Q

Words that are palindromic…

A

Level

Madam

Rotator

Deleveled

39
Q

Discuss the probability of restriction site occurring in DNA Fragment

A

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

40
Q

Explain the functioning of DNA ligase

A
  • DNA joining enzyme called DNA ligase
  • Catalyses the formation of phosphodiester bond
  • 5’ phosohate of one nucleotide to the 3’ hydroxyl of the next nucleotideRestriction enzyme recognizes siteCleaves phosphodiester bondsBUT 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)
41
Q

How is restriction and ligation used to clone ?

A
  • 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

42
Q

Describe modification of blunt ends

A

-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

43
Q

What are the types of nucleases?

A

Endonucleases- internal cuts

Exonuclease- nucleotides removed from the ends

44
Q

Description cleavage sites

A
  • 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
45
Q

Explain restriction mapping of recombinant plasmid to determine fragment orientation

A

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
46
Q

Describe restriction mapping in vector

A

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
47
Q

Restriction endonucleases are often…

A

Homodimer

Sliding/jumping until coupling occurs,

Catalysis leads to enzyme being released and DNA cut

48
Q

Explain restriction mapping of recombinant plasmid to determine fragment orientation

A
  • 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

Molecular Biology (24 decks)

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