Biology- Chapter 12: DNA Technology and Genomics

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


1. mixtures of restriction fragments belonging to the same individual


2. 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