1
Q
Recombinant DNA technology
A
- a set of molecular techniques for locating, altering and studying DNA segments
- also referred to as genetic engineering
- now encompasses many molecular techniques that can be used to analyze, alter and recombine virtually any DNA sequence from a number of sources
2
Q
Restriction enzymes
A
- part of bacterial defense systems
- enzymes that cleaver around or within a specific DNA site
- recognize and make double-stranded cuts in DNA at specific nucleotides sequences (type II used most)
- some make staggered cuts, while others make blunt cuts
3
Q
cohesive or sticky ends
A
- DNA fragments with short, single-stranded overhanging ends
- they are complementary to each other and can spontaneously pair to connect the fragments
4
Q
DNA ligase
A
joins together the two DNA fragments by sealing the sugar-phosphate backbone
5
Q
electrophoresis
A
separates DNA or RNA based on size, or proteins based on size and charge.
6
Q
gel electrophoresis
A
- a porous gel is often made of agarose which is melted and poured into a plastic mold. the gel solidifies as it cools.
- DNA samples loaded into small wells made at one end of the gel and an electrical current is run through it
- DNA carries a negative charge and migrates toward the positive pole. The gel separates DNA fragments by size, with small migrating faster
- A standard with known DNA fragments included for comparison to the DNA sample, and the fragments are visualized with a dye such as ethidium bromide which fluoresces under UV light
7
Q
probes
A
- DNA or RNA molecules with sequence complementarity to the gene of interest.
- radioactively or chemically labeled for detection.
- used to identify specific fragments of DNA
8
Q
Southern blots
A
- developed from a man’s name
- used to detect DNA separated by fragment length
9
Q
Northern blots
A
- used to detect RNA separated by length
10
Q
Western blots
A
- used to detect proteins separated by charge, weight, etc
- probes are antibodies.
11
Q
Gene cloning
A
- the production of identical copies (clones) of an original piece of DNA.
- Accomplished by inserting DNA fragments into bacteria so that they will be stable and copied by the bacteria.
12
Q
Cloning vector
A
- stable, replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into a cell
- carry DNA of interest
- carry be prokaryotic or eukaryotic
13
Q
features of cloning vectors
A
- origin of replication
- selectable marker
- one or more unique restriction enzyme sites
14
Q
plasmid vectors
A
commonly used vectors for cloning genes in bacteria
15
Q
Cloning Genes
A
- DNA fragment can be inserted into a plasmid vector by cutting the foreign DNA and the plasmid with the same restriction enzyme
- if the enzyme makes staggered cuts in the DNA, complementary sticky ends are produced in the foreign and plasmid DNAs
- the DNA and the plasmid are then mixed together; some of the foreign DNA will pair with the cut ends of the plasmid. Alternatively, no foreign DNA is inserted and the plasmid ends with base pair with each other, creating the original plasmid vector.
- DNA ligase used to seal the nicks in the sugar-phosphate backbone
- once DNA fragmented cloned into the vector, they are introduced plasmids into bacteria so they can be replicated through transformation
16
Q
transformation
A
bacterial cells take up DNA from the external environment. Some bacteria undergo transformation naturally; others must be treated chemically or physically before they will undergo transformation.
17
Q
antibiotic resistance genes
A
confer resistance to antibiotics that normally kill bacteria. Any cell that contains a plasmid with such a marker will be able to live in the presence of the antibiotic.
- Amp^r gene confers resistance to ampicillin
18
Q
LacZ and blue/white screening
A
- The lacZ gene encoding B-galactosidase (B-gal) is used to screen for insertion of foreign DNA
- Insertion of DNA into the plasmid interrupts the lacZ gene, and no B-gal produced
- B-gal can cleave an analog of lactose called X-gal which turns bacteria blue when cleaved.
- Bacteria transformed with the plasmids are plated on medium containing ampicillin and X-gal
- if the lacZ gene not present or has been interrupted, then the bacterial colony will appear white
- Only bacteria that have received ampR plasmid will form a colony on the plate. If the cell carries a recombinant plasmid (foreign DNA inserted into lacZ), colonies are white; if no DNA inserted, colonies are blue.
19
Q
expression vectors
A
- contain sequences required for transcription and translation in bacterial cells in addition to the usual elements
20
Q
eukaryotic cloning systems
A
- many eukaryotic proteins are modified after translation by the addition of a carbohydrate group. essential for function but can’t be carried out in bacteria
- cloning vectors developed for insertion of genes into eukaryotic cells
21
Q
yeast artificial chromosome
A
- DNA molecule with a yeast origin of replication, a pair of telomeres and a centromere.
- stable, replicate and segregate in the same way as yeast chromosomes
- carry DNA fragments as large as 600 kb and have been modified so they can be used in eukaryotic organisms other than yeast
22
Q
Ti plasmid
A
- vectors from this plasmid have been used to transfer genes that confer economically significant attributes such as resistances to herbicides, plant viruses and insect pests.
23
Q
PCR
A
- polymerase chain reaction
- method for rapidly amplifying a DNA sequence that might constitute a tiny fraction of a DNA sample or be very rare.
- basis of PCR is replication catalyzed by a DNA polymerase
24
Q
requirements for PCR
A
- single-stranded DNA template from which a new DNA strand can be copied
- a pair of DNA primers with a 3’ OH group to which new nucleotides can be added.
25
Steps in PCR
- heat sample to 90-100 degrees to break hydrogen bonds and separate DNA into two templates
- cool sample to 30-65 degrees to allow complementary nucleotide primers to anneal to the DNA template
- heat sample to 60-70 degrees for elongation of the primers with a thermostable polymerase that can remain active at high temps and replication
- repeat cycle
26
result of PCR
with each cycle, the amount of DNA doubles. One DNA molecule increases to more than 1000 in 10 cycles and to more than 1 billion in 30 cycles.
27
Reaction components of PCR
- target DNA
- DNA polymerase
- 4 dNTPs
- primers
28
Taq polymerase
- incredible stable at high temperatures and is not denatured in the strand-separation step of PCR
29
How do we find a gene sequence?
Clone first and search later
30
Shotgun cloning technique
clone a large number of DNA fragments and then search for the fragment of interest among the clones.
31
genomic DNA library
- contains of all the DNA sequences found in an organism's genome.
- must contain a large number of clones to ensure that all DNA sequences are represented
32
cDNA libraries
- An alternative to a genomic library is a cDNA library that consists only of those sequences transcribed into mRNA
33
in situ hybridization
- used to determine the location of genes or their expression
- DNA or RNA visualized while it is still in the cell
- for chromosomes, the cells must be fixed and the chromosomes must be spread on a slide and denatured.
- A labeled probe is then applied, just as it would for a Southern blot
34
Sanger sequencing (Chain-Termination DNA Sequencing)
- dideoxy sequencing
- requires ssDNA template, DNA primer, DNA polymerase, radioactively or fluorescently labeled nucleotides and modified nucleotides (dideoxys)
- The fragment of DNA to be sequenced is used as a template to make a series of new DNA molecules
- DNA sample divided into four separate sequencing reaction, containing four standard dNTPS and the DNA polymerase. To each reaction is added only one of four ddNTPS
- After a ddNTP incorporated into DNA strand, no more nucleotides can be added, because there is no 3' -OH group to form a phosphodiester bond with an incoming nucleotide
- ddNTPs terminate DNA synthesis by producing DNA strands of different lengths, each of which ends in the same base.
35
the sequencing region
- copes of the target DNA are isolated and split into four tubes.
Each tube contains:
- many copies of a primer complementary to one end of the DNA target strand
- all four types of dNTPs
- a small amount of one of the four types of ddNTPs
DNA polymerase
36
Automated sequencing
- the dideoxy method is used but ddNTPs are labeled with fluorescent dyes; a different colored dye is used for each type.
- the four sequencing reactions are done in the same tube, and electrophoresed in one capillary tube containing the gel
- the different-size fragments separated within a tube migrate past a laser beam and detector
- as the fragments pass the laser, their dyes are activated and the fluorescence is detected by an optical scanner.
37
DNA fingerprinting
- the use of DNA sequences to identify individual person.
- uses micro satellites or short tandem repeats
- basically each person has a different number of short tandem repeats and when amplified you can compare the amount of STRs per person to the DNA found
38
STRs
- very short DNA sequences repeated in tandem
- found at many loci in the human genome and people vary in the number of copies of the repeats at each locus
- typically detected by PCR. The length of the amplified fragment depends on the number of repeats.
39
forward genetics
- begins with a phenotype (a mutant individual) and proceeds to a gene that encodes the phenotype
- relies on the identification and isolation of random mutations that affect a phenotype
40
Reverse genetics
- begins with a genotype - a DNA sequence - and proceeds to the phenotype by altering the sequence of inhibiting its expression.
- relies on the ability to create mutations, not at random, but in particular DNA sequences, and then to study the effects of these mutations on the organisms.
41
transgenic organism
an organism that has been permanently altered by the addition of a DNA sequence
42
transgene
the foreign gene
43
knock out
- variation of the reverse transgene approach
| - fully disable a gene to determine the effect on phenotype
44
blunt ends
cleavage is straight through the DNA
45
origin of replication
- ensures plasmid is replicated in bacterial cell
46
cloning site
- the site to which DNA of interest will be inserted
47
selectable marker
- help determine if plasmids are successfully transformed
48
purpose of dideoxy sequencing
They stop synthesis at a specific site, so the base at that site can be determined.
GN 311
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