Molecular Genetics (Unit 3) - chapter 8 Flashcards
______ is the intentional production of new genes
and alteration of genomes by the substitution or introduction of new genetic material
Genetic engineering
_______ Is a fragment of DNA composed of two sequences originating from at least two different sources
Recombinant DNA
In order to form recombinant DNA, a molecular biologist
requires tools to ____, ____, and ____ DNA
cut, join, and replicate
These tool include:
Restriction enzymes
DNA ligase
Plasmids
An enzyme that cuts DNA at specific base pair sequence
Restriction Enzymes
Each type of restriction enzyme is a able to recognize a
specific sequence of ______ that is known as its
_______ site
nucleotides
recognition
A fragment that is produced when a DNA strand is cut by
these enzymes is called a ________
restriction fragment
Restriction enzymes are
biological molecules, which are
isolated from _______
bacteria
The function of these enzymes in bacteria is to:
cut foreign viral DNA into pieces
Two types of cuts are possible
______ ends occur when cuts are
made straight across the DNA
_____ ends occurs when cuts are
made in a zigzag across the DNA
Blunt
Sticky
_______ an enzyme that is used for “reassembly” of DNA
sticky ends
DNA ligase
__ DNA ligase works well with blunt ends
T4
A desired gene must be inserted into a _____ location
specific
______ are small, circular pieces of DNA that are found in
bacteria
Plasmids
DNA ligase Joins ______ bonds together
phosphodiester
A plasmid that has been designed to transfer foreign genetic material into a cell is called a _____
vector
The same restriction enzyme is used both on the source DNA
to __________ and the plasmid so that they will have
the same sticky ends.
isolate target DNA
Cloning DNA fragments proccess diagram:
Bacteria are able to uptake a plasmids under specific
conditions The introduction of DNA from another source is
known as ______
transformation
_______ or commonly called plasmid maps are a
diagram that illustrates restriction enzyme recognition sites and the distances, measured in base pairs, between sites.
Restriction maps
is a technique used to make a large number of copies of a DNA sequence.
Polymerase Chain Reaction (PCR)
There are three steps to PCR:
Denaturation
Annealing
Elongation
______- Cool the mixture to 50-65oC to allow primers to
anneal to the DNA
Annealing
_______ – The DNA target sequence is heated to
94-96oC to denature it into single strands
Denaturation
Millions of copies of the _________ can be produced in two hours.
DNA sequence
________ – Heat to 72oC for the DNA polymerase to extend
strand
Annealing
The cycle is ______ several times. After the _____ cycle, two of the eight match target sequence.
repeated
third
A method for separating large molecules such as DNA, RNA,
and proteins
Gel Electrophoresis
Gel electrophoresis separates DNA fragments based on their
_____ and ______ properties
chemical and physical
DNA fragments migrate through the gel at a rate that is
inversely proportional to their ___. The smaller the fragment
is, the ______ it will travel through the gel.
size
faster
To be able to see the DNA fragments after the gel has been run, _______ is used to stain the gel.
ethidium bromide
________ is a process in which the sequence of a
strand of DNA is determined
DNA sequencing
Uses of dna sequencing:
for understanding how a gene functions, mutations,
locate regulatory sequences and compare homologous genes of
different species
understand Chain Termination (Sanger
Method)
-Developed by Fredrick Sanger in 1970’s
-It relies on the addition of a labelled dideoxynucleotide
(ddNTP) to a growing DNA strand
-ddNTPs prevent any further binding of nucleotides
-The labels on the ddNTPs are dyes that fluoresce and can be
used to identify the specific base when exposed to the laser
light
-Four reaction tubes are set up, each with many normal
nucleotides (dATP, dTTP, dGTP, dCTP), multiple copies of
the DNA to be sequenced, DNA polymerase, a DNA primer).
-Only one of the labelled dideoxynucleotides (ddATP, ddTTTP,
ddGTP, and ddCTP) are added each tube
-This produces fragments of different sizes, each with their last
nucleotide labelled
Sequencing the Results:
The contents of the four tubes are combined and separated by
gel electrophoresis
The sequence can then be determined by reading the order of
the labelling type that corresponds to a specific ddNTP
This process is now computerized (fig 5 p.381)
Whole Genome Shotgun
method:
Involves producing many DNA fragments using a pressurized
syringe
Then uses Sanger sequencing and computers to sequence and
reassemble the fragments (fig 6 p.381)
Drawback of this method is for repeating segments- its hard to
detect where these segments are repeated. This sometimes
causes some errors in the results.
Analyzing Genomes:
Once the DNA has been sequenced it can be analyzed for its
actual sequence (structural genomics) and/or for its function
(functional genomics)
Most research is focused on functional genomics and it uses
bioinformatics
Bioinformatics uses both lab experimental data and
computerized analysis
Nanopore Sequencing:
Individual strands of DNA through microscopic holes called
nanopores
The sequence of the DNA is read as it passes through the pore
by the unique current each bases conducts as it passes through
the silicon sheet
mRNA is isolated from a reference cell (normal) and an
experimental cell (eg. cancer cell)
Used to build complementary (cDNA) libraries
cDNA sequences are labelled with fluorescent dyes
Green for normal cDNA
Red for experimental cDNA
cDNA strands are denatured into single strands, then placed on
microarray
DNA microarrays:
Helps scientists pinpoint the functions of specific genes
Microarray or gene chip, is designed to hold many individual
DNA samples
Samples are called probes – known sequences of genes for the
organism/cell under study
Location of each probe on the gene chip is also known
cDNA will hybridize to any probes on the gene chip that they
share a complementary sequence with
Any non-complementary sequences will be washed off the
chip
Fluorescence is detected and analyzed
Green spots mean normal cells express that gene
Red spots mean experimental cells express that gene
Yellow spots mean both cells express that gene
:. can determine which genes have altered expression in the
experimental cells
Advantageous because thousands of genes can be analyzed at
the same time
