gene tech Flashcards
genome
complete set of genes in a cell. can include:
- genes coding for proteins
- regulatory genes (ie: those coding for TF, siRNA/miRNA)
- mitochondrial dna
- chloroplast dna
- genes coding for antibiotic resistance located in plasmids
- viral RNA/DNA in capsid of virus
PHYLOGENETICS
automated sequencing of genomes of species; comparing DNA base sequences and amino acid sequences
proteome
full range of proteins a cell can produce
recombinant/transgenic dna
cell having two or more sources of dna
describe how dna is replicated inside of a cell
dna strands separate
parent strand acts as template
complementary base pairing
dna polymerase joins adjacent nucleotides via phosphodiester bonds
consensation reaction
5’ to 3’ direction
semi conservative replication formed by
each new molecule has 1 template and 1 new strand
describe how the polymerase chain reaction (PCR) is used to amplify a dna fragment
taq dna polymerase
add nucloetodies and primers
heat to 95 degrees to break H bonds and seprate strands
reduce temp to 55 degrees so primers bind to dna strand
increase temp to 70 so dna polymerase can add the nucleotides
why is the dna heat to 95 degrees during PCR`
produces single strand dna
breaks weak hydrogen bonds between strands
why do you add primers during PCR
complementary to end of fragment
replication of base sequence (from there)
prevents strands annealing
isolating dna fragments
mrna converted to cdna via reverse transcriptase
use restriction enzymes to cut a fragment
create the gene in a gene machine
dna polymerase makes dsCDNA
advantages of reverse transcriptase
mrna easier to obtain
introns have been removed, exons spliced together
bacterial dna doesnt contain introns
restriction endonucleases
hydrolyse dna at specific recognition base sequences
- complementary to enzyme active site
- recognition sites are palindromic, base pair read the same in opposite directions
what will happen if the recognition sequences for the restriction endonucleases occurs within the dna fragment you want to isolate
will cut this gene and it will not code for a functional protein
cuts are either blunt/sticky end
blunt ends
used in pcr and gel electropheresis
sticky ends
used in transformation
gene machine
- desired nucleotide sequence fed into the computer
- synthesis of short sequences of nucleotides (oligonucleotides)
- loigonucleotides overlapped and made ds via PCR
- ## gene inserted into bacterial plasmid
benefits of using gene machine
dna without introns
faster than enzyme controlled reactions
artificial genes easily transcribed and translated by prokaryotes
vector
dna carrier (ie: virus, plasmid of bacteia) used to transfer forign dna into cells
insertion of genes via vector
isolated target dna fragment inserted into vector dna by cutting open vector dna using SAME restriction endonucleases to produce specifically complementary “sticky ends”
- complementary base pairing of sticky ends between the vector dna and target fragment dna
- dna ligase joins dna fragment and vector dna (ligation via phophodiester bonds)
- new recomnincant dna
outline a method for in vivo cloning
vivo = living
- cut desired gene from organism via restriction endonucleases
OR use mRNA from cell via reverse transcriptase to form cDNA
THEN
- make artificial dna with correct sequence of bases
- using dna polymerase
-cut plasmid open
- with same restriction endonucleases
- sticky ends complementary to each other and attach
- dna ligase joins
- return plasmid to bacterial cells
marker gene
allows easy identification of cells that have taken up genetically transformed plasmid
common types:
- fluorescence
- antibiotic resistance
- enzyme markers
indeitfying transormed bacteria using antibiotic resistance genes
no plasmid = killed by both antibiotics
original plasmid = resistant to both antibiotics
transformed plasmid taken up = resistant to one antibiotic but not second (2nd cut and disrupted by inserting foreign dna)
primers
short pieces of ss dna with complemenary base sequences atg start of dna fragment. prevent dna strands sticking to each other.
describe the process of pcr
heat dna to 95 degrees breaks weak hydrogen bonds
- add primers and add nucleotides
- cool to 50C to allow binding of primers/nucleotides
- add taq dna polymerase
heat to 75C
- dna polymerase joins nucleotides together
- repeat cycle many times
formula for number of dna strands after x number of cycles
2^n where n = number of cycles
no of cycles = log2 (no of dna molecules)
why does the exponential curve increase in dna molecules start to plateaue after 35 cycles?
low conc. of nucleotides and primers
dna polymerase cant bind to nucleotides
SO less new strands of DNA formed as dna polymerase needs primers to start strand synthesis
explain why base pairs is a suitable unit for measuring the length of a dna piece
dna = 2 chains
AT, CG
- bases are a constant distance apart
- each base pair is the same length
a deletion mutation occurs in gene 1. describe how a deletion mutation alters the structure of a gene.
- removal of one or more nucleotides
-frameshift
describe the main stages in the copying, cutting and separation of the dna
heat dna to 95C
strands separate
cool so primers bind to dna
add dna taq polymerase
restriction enzymes cut dna at specific base sequences
use of electric current/gel
shorter fragments move further
describe a plasmid
circular dna
separate fro main bacterial dna
contains only a few genes
suggest one reason why dna replication stops in the pcr
limited number of primers
dna polumerase denatures eventually
suggest why the restriction enzyme has cut the hujman dna in many places and the plasmid dna only once
- enzymes only cut at specific base sequences
- occurs once in plasmid and many times in dna humans
describe how the bacteria containing the insulin gene is used to obtain sufficient insulin for commercial use
- use of fermenters
- provides nutrients
- named environmental factor
- reproduction of bacteria
- insulin accumulates and is extracted
explain what is meant by a vector
carrier of dna
into cell/organism
in vivo cloning vs in vitro cloning
vivo: vitro
slower, one dna rep per cd: quicker, millions off copies
dna/rna/proteins modified, introns removed: no modification
corredcting error mechanisms, more accurate: lacks error correcting method
benefits of recombinant dna
develop medical applications (spread insulin)
- aricultural applications (plants resistant to disease and weather)
viruses as vectors
can cause immune response and formation of cytotoxic t cells
germ line therapy cons
DNA transfer to cells producing gametes
- imperfect solution
- denial of human rights, no consent
- abuse, favourable characteristics
somatic gene therapy cons
dna transfer to normal tissue
- not all cells take up recombnant dna
- not all cells xpress dna allele
- only some tissue types are accessible
- multiple treatments
- immune response to vector
explain how modified plasmids are amde from genetic engineering and hjow the use of the markers enable bacteria containing these plasmids to be detected.
isolate target gene from organism via restriction endonucleases/ rna from otger cell using reverse transcriptiase
- get dna to produce sticky ends
use dna ligase to join taret dna gene to plasmid
also include markwer gene (ie: antibiotic resistance)
- add plasmid yo bacteria to grow
bacteria expressing marker gene wll have target gene too
mrna may be described as a polymer. explain why.
made of of many similar monomers
what is a dna probe
short ss of dna
bases complementary with dna/allele fragment to be located
radioactive/labelled by fluourescence.
name 3 techniques used by scientists to compare dna sequences
- pcr
- dna fingerprinting
-gel electropheresis
dna hybridisaton
dna probe binds to complementary target dna
- positive=
- crrier for recessive allele for genetic disorders
- mutation in known genes linked to increase risk of a disease.
vntrs
variable number tandem repeats
- region of dna between genes
- differences in vntrs can be analysed via gel electropheresis
describe the process of dna fingerprinting
- dna extracted from sample
- dna hydrolysed by restriction endonucleases
- produces blunt ends, cut outside of vntrs to leave intact
- separate fragments according to size via gel electropheresis
- by putting dna in a well and applying current
- make fragments ss by putting in strong alkali
- add radioactive gene probes to allow positions of fragmenrs to be visualised as bands
- put nylon on top fix ss dna to nylon with uv light
- identify base sequence of interest using x ray film (autoradiography)
