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Flashcards in essential tools in molecular biology Deck (9)
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1
Q

define gene cloning and discuss the process (removing a gene and manipulating it)

A

1)identifying a gene

2) isolating and extracting a gene
a) lyse cell with a detergent to disrupt the plasma membrane
b) apply proteases and Rnases to destroy metarial that would make gene DNA less pure
c) centrifuge sample to pellet debris. supernatent quality can be checked using spectrophotometry
d) use mechanical sheers to remove piece of DNA

3) modifying the gene in test tubes with different enzymes; nucleases, ligases, polymerases, modifying enzymes, topoisomerases
4) reintroducing the gene into another organism

2
Q

discuss the 5 classes of enzymes used to modify DNA; 17 marks

A
1) nucleases; break the phosphodiester bond in DNA which breaks DNA into fragments or degrades DNA
endonuclease/exonuclease classification
class 1, 2 and 3 classification
class 1; recognise a specific sequence but have a non specific cleavage site
class 2 (restriction enzymes); have a very specific sequence site and cleavage site allowing DNA to be cut precisely
class 3; have methylase activity as well as nuclease activity

2)ligases; bond pieces of DNA together by reforming the phosphodiester bond
functions in nature include repairing breaks in DNA, joing pieces of DNA together and joining a piece of DNA into a circular piece of DNA
if sticky ends are present ligation is more efficient because the strand form hydrogen bonds with eachother and give the ligase enzyme something to work on

3) polymerases; enzymes that synthesise new DNA strands from preexisting DNA strands
a) DNApol1 (DNA-DNA); 5’ to 3’ synthesis, 5’ to 3’ endonuclease (remove DNA infront of current position) and 3’ to 5’ endonuclease (remove errors)
b) Klemow fragment DNApol (DNA-DNA); can only synthesise from ssDNA (its ised for cDNA and sequencing). can do 3’ to 5’ nuclease activity)
c) reverse transcriptase (RNA-DNA); 5’ to 3’ synthesis. is used to make cDNA. can do rna exonuclease in both directions
d) taqDNApol (DNA-DNA); 5’ to 3’ synthesis with no exonuclease activity so no proofreading ability. very quick and not denatured at high temperatures so is used for PCR

4) modifying enzymes; add or remove chemical groups
a) alkaline phosphatases; remove the 5’ phospate; this stops plasmids recircularising without their GOI
b) terminal deoxynucleotidyl transferase; adds a single deoxynucleotide on the 3’ end of a DNA strand
c) polynucleotide kinase; adds a phosphate to the 5’ end end of DNA

5)topoisomerases; adds or removes supercoils from circular DNA.

3
Q

how are restriction enzymes named; 3 marks

A

first 3 letters come from genus of origin
fourth letter represent species of origin
the number is the number of the restriction enzyme
EcoRIII; Escherichia coli’s 3rd restriction enzyme

4
Q

define the terms prototype, isochizomers and neoschizomer

A

prototype; first enzyme discrovered which recognises a specific sequence
isochizomer; an enzyme discovered after the prototype which recognises the same sequence
neoschizomer;an enzyme discovered after the prototype which recognises the same sequence but has a different cleavage site

5
Q

discuss gel electrophoressis. 3 marks

discuss the three types of gel electrophoresis

A

a method of seperating a sample of proteins based on their size
DNA is first placed in a gel with a labeled dye such as ethidium bromide. gel is placed in a salt solution. DNA is negatively charged so travels to a positive cathode.
the gel has pores in it so large molecules move slower than small molecules

1) polyacrylamide gel; accurate but size limited (2kb)
2) agarose gel; not accurate but not size limited (10kb max)
3) pulsed field; electric field changed periodically; allows for very large molecules to be seperated (better resolving power)

6
Q

name 3 modern gene editing technologies; 3 marks
discuss how these technologies work in general and specifically how one works; 4 marks
name 2 examples of one method being used

A

CRISPR-Cas9, Zinc finger nucleases, TALENS

create double strand breaks in DNA at precise locations, the ends can then rejoin (knock out).
multiple cuts can be done at once quickly
knock in applications exist; if the ds break is made a new piece of DNA can be inserted into the DNA

CRISPR-Cas9
a piece of guide RNA (gRNA) binds to a specific sequence of DNA and guides Cas9 (endonuclease) to the sequence where it cuts the DNA just upstream of a PAM motif
the Cas9 is guided by the trascRNA region of gRNA

the myostatin gene would normally inhibit muscle development in cattle. this gene was knocked out which led to increased muscle growth

removing the gene for horns in cattle so that mothers arent harmed during birth

7
Q

discuss nuclear transfer experiments dont in the 1950’s and what we learnt from them; 4 marks

discuss how dolly relates to these findings; 4 marks

discuss problems associated with dolly and this type of nuclear transfer

A

nuclear transfer was done to gastrula cells and post gastrula cells (egg harvested and had its nucleus killed with UV light before inserting another nucleus into the egg)
development was successful in gastrula cells but not in post gastrula cells
as cells because more differentiated and specialised they lose their flexibility and cant become unspecialised

dolly was made by nuclear transfer using a fully differentiated egg cell and inserting a whole other cell rather than just the nucleus. (electrical fusion caused the two cytoplasms to join)
successful development occurred which was interesting because previously people had thought it impossible to do this with a fully differentiated cell
the offspring was genetically identical to dolly

opened up debate about cloning humans; simply take some of their cells and add their nucleus to an egg cell

problems

1) very inefficient; 277 cell fusions were done before a successful one occured
2) offspring abnormalities are common such as large offspring syndrome in which large offspring damage the mother
3) epigenetic effects mean the animal isint an exact replica

8
Q

is nuclear transfer defined as transgenesis

A

no

37 genes from donor oocyte are incorporated (mitochondria genes for example)

9
Q

discuss applications of reproductive cloning

A

1) production of elite animals which can then be bred with other animals. its very expensive; £80,000 for a horse
2) cloned pets; barbra stressand
3) perform nuclear transfer on pigs so that their hearts have different antigens and can be better used for organ donation