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What is the average amount of DNA that is similar between humans?
99.9%
How much of human’s DNA is noncoding?
97%
What is noncoding DNA made up of?
gene control sequences (ex: promoters)
introns
DNA located between repetitive DNA sequences
Where are the repeated DNA sequences found?
at the centromeres and the end of the chromosomes
What are the steps in DNA identification?
copying DNA
cutting DNA
sorting DNA by size
comparing DNA
What method is used when the source of DNA is scanty, impure, or in a partially degraded state?
the polymerase chain reaction (PCR) to prepare large quantities of a particular gene
What does the PCR method involve?
using short DNA sequences called primers to select the portion of the genome to be amplified
What are automated PCR machines called?
thermocyclers
how many molecules can the thermocycler produce in a few hours?
100 billion
PCR cannot replace gene cloning in cells when ___ ?
large amounts of DNA are needed
What is the DNA sample mixed with?
a heat tolerant DNA replication enzyme (DNA polymerase), DNA nucleotides, and primers that are complementary to the ends of the DNA fragment that is to be copied.
What is needed to start replication?
primers
This solution is exposed to what?
cycles of heating (to separate the DNA strands) and cooling
What enzyme can withstand the heat of each cycle?
the unusual DNA polymerase
How do scientists generate millions of copies of a single DNA fragment?
they choose a DNA fragment to copy and design primers that will bind to both ends of the fragment. DNA polymerase copies the segment between the two primers.
What are restriction enzymes?
they cut DNA at specific sequences
How long of a DNA sequence do the restriction fragments recognize?
4-8 nucleotides long
What are restriction fragments?
pieces of DNA produced from the restriction enzymes cutting it
What are “sticky ends”
2 double-stranded DNA fragments with single- stranded overhanging ends
Sticky ends DNA restriction fragments from ?
different sources
How do you add a piece of DNA from another source?
by cutting it using the same restriction enzyme
What does gel electrophoresis do?
it sorts DNA molecules by size
How does gel electrophoresis separate nucleic acids or proteins?
it uses a gel (a thin slab of jellylike material) as a molecular sieve
On what basis are the nucleic acids or proteins seperated?
size or electrical charge
How many steps are there to separate DNA in different mixtures?
7
What is the first step?
Restriction enzymes are used to prepare DNA fragments in each mixture
What is the second step?
A sample of each mixture is placed in a well at one end of a flat, rectangular agar gel slab
What is the third step?
A negatively charged electrode from a power supply is attached near the DNA-containing end of the gel, and a positive electrode is attached near the other end.
What is the fourth step?
the DNA molecules all travel through the gel toward the positive pole.
Are DNA molecules negatively charged?
yes
because of their phosphate groups
What is the fifth step?
As they move, the polymer fibers within the gel slows down the movement of the longer molecules more than it does shorter ones, separating them by length.
What is the sixth step?
After about 1⁄2 hour the electrodes are disconnected
What is the seventh step?
gel electrophoresis separates a mixture of DNA molecules into bands
Are these bands of DNA the same length?
yes
the shorter molecules are towards the bottom
What are restriction fragment length polymorphisms (RFLPs)?
The differences in restriction fragments produced in this way
How many base pairs are different between two people?
3 million
What does electrophoresis allow us to see?
Similarities as well as differences between
mixtures of restriction fragments belonging to the same individual
the base sequences in DNA from two individuals.
How do you permanently preserve DNA fragments that are isolated by gel electrophoresis?
the pieces of DNA are transferred or ‘blotted’ out of the fragile gel onto a nylon membrane
How many steps are there to make a DNA fingerprint?
5
What is the first step?
A positively charged nylon membrane is placed over the gel and the negatively charged DNA fragments are transferred to membrane
What is the second step?
DNA is then ‘unzipped’ to produce single strands of DNA
What is the third step?
Biologists incubate the nylon membrane with radioactive probes. They can prepare a nucleic acid probe complementary to the DNA of interest and label it radioactively.
What is the fourth step?
A sheet of X-ray film placed over the gel will be exposed only where the desired DNA is on the gel.
What is a fingerprint?
The resulting pattern of bands
What is the fifth step?
To compare two or more different DNA fingerprints the different DNA samples are run side-by-side on the same electrophoresis gel
What are probes?
small fragments of minisatellite DNA tagged with radioactive phosphorous
When can the information be used to synthesize a short single strand of DNA with the complementary sequence?
When at least part of the nucleotide sequence of a gene is already known or can be guessed
What is the DNA labeled with?
a radioactive isotope or fluorescent dye
What is a nucleic acid probe?
This labeled, complementary single-stranded nucleic acid molecule
What is n.a.p. used for?
to find a specific gene or other nucleotide sequence within a mass of DNA
What do the probes hydrogen bond to?
the complementary sequence in the targeted DNA
What is this method used for?
detecting genes/specific DNA pieces depend on base pairing between the gene/DNA piece and a complementary single strand sequence on another nucleic acid molecule, either DNA or RNA.
What can happen once the researcher identifies a colony carrying the desired gene?
the cells can be grown further and the gene of interest (and/or its protein product) isolated in large amounts
What are minisatellites?
short sequences (10-60 base pairs long) of repetitive DNA that show greater variation from one person to the next than other parts of the genome
What is this variation exhibited in?
stutters or VNTRs
How many VNTR loci does DNA fingerprinting usually compare?
5-13
DNA fingerprinting simultaneously does what?
detects lots of minisatellites in the genome to produce a pattern unique to an individual
What are the odds that 2 people will share a DNA profile produced by 13 VNTR loci?
1 in a 100 billion
What does VNTR stand for?
variable number tandem repeats
What are length polymorphisms?
The non-coding DNA segments that vary in their length
What are some length polymorphisms made out of?
short repeating sequences of DNA nucleotides
What are VNTRs?
The nucleotides that repeat few or many times in tandem (one after another) in length polymorphisms
Is the amount of tandam repeats in a specific loci in DNA the same for everyone?
no, that’s why it is used for DNA identification
What does recombinant DNA technology do?
combines genes from different sources–even different species–into a single DNA molecule
How do researchers create recombinant DNA?
by inserting desired genes into plasmids
What are plasmids?
small, circular DNA molecules that replicate separately from the much larger bacterial chromosome
Are plasmids required for cell reproduction or growth?
no
What genes do plasmids carry?
genes that cause the bacterium to cause diseases or provide resistance against antibiotics.
What happens when DNA from another source is introduced to the plasmid?
they hydrogen bond with the plasmid DNA
How are the bonds made permanent?
DNA ligase forms covalent bonds between adjacent nucleotides
How many times is the plasmid cleaved?
one
the restriction enzymes creates sticky ends on what?
both the human DNA fragments and the plasmid
What is transformation?
A process by which bacteria take up DNA from the surrounding
The bacteria reproduces to produce what?
a clone of cells
How can cloned genes be used?
directly or to manufacture protein products
What is a cloning vector?
they copy genes and move them from one organism to the next
Can plasmids be used as cloning vectors?
yes
the restrction enzyme makes ___ of human DNA fragments
millions
What is the genomic library?
The entire collection of all the cloned DNA fragments from a genome
What are vectors?
short pieces of DNA that are capable of replicating on their own when inside a cell
they’re used for cloning
can be bacterial plasmids or phages
How is a phage used?
The DNA fragments are inserted into phage DNA molecules. The recombinant phage DNA can then be introduced into a bacterial cell through the normal infection process.
What happens to the phage inside the cell?
The phage DNA replicates and produces new phage particles, each carrying the foreign DNA. A collection of phage clones can constitute a second type of genomic library.
How do they identify the clone with the desired gene?
with testing the protein product or
viewing it under UV light or exposing it to photographic film, the probe glows and reveals its location.
How does reverse transcriptase help make cloned genes?
it makes complementary DNA which is shorter due to the lack of introns
What are cDNA libraries used for?
studying genes responsible for specific functions in a specific cell
What is the Human Genome Project?
an effort to map the human genome in total detail by determining the entire nucleotide sequence of human DNA
What are bacteria used for?
to manufacture protein products on a large scale because plasmids and phages are readily available and can be grown rapidly and cheaply
What are sheep cells used for?
they add glycoproteins to the initial ones made in bacteria
What is a GMO?
an organism that has acquired 1 or more genes by artificial means rather than through normal breeding methods
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