Lecture 15 Molecular Genetic Analysis and Biotechnology

Question 1

Southern blot =
Northern blot =
Western blot =

Answer 1

DNA
RNA
Protein

Question 2

Probe:

Answer 2

DNA or RNA with a base sequence complementary to a sequence in the gene of interest

Question 3

Gene cloning:

Answer 3

Amplifying a specific piece of DNA via a bacteria cell

Question 4

Cloning vector:

Answer 4

a replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell

Question 5

What are some of the effects and practical applications of molecular genetic analysis?

Answer 5

Molecular genetics has had profound effects on all fields of biology. Whole genomes have been sequenced, structures of genes elucidated, the patterns of molecular evolution studied. Recombinant DNA technology is now used to diagnose and screen for genetic diseases, and gene therapy is being explored. Recombinant DNA is used to make pharmaceutical products, such as recombinant insulin and clotting factors. Genetically modified organisms is changing the lives of farmers and improving agricultural productivity and the quality of food and fiber.

Question 6

What feature is commonly seen in the sequences recognized by type II restriction enzymes?

Answer 6

The recognition sequences are palindromic, and 4-8 base pairs long

Question 7

What are restriction enzymes?

Answer 7

Enzymes that recognize and cut DNA at specific nucleotide seqences

Question 8

What normal role do restriction enzymes play in bacteria?

Answer 8

Restriction enzymes cut foreign DNA, such as viral DNA, into fragments.

Question 9

How do bacteria protect their own DNA from the action of restriction enzymes?

Answer 9

They modify bases, usually by methylation, at the recognition sites

Question 10

What are cohesive ends?

Answer 10

Fragments with short, single-stranded overhanging ends

Question 11

What are blunt ends?

Answer 11

Even-length ends from both single strands

Question 12

What ends are harder to rejoin?

Answer 12

blunt ends

Question 13

Which ends are sticky?

Answer 13

cohesive ends

Question 14

How is gel electrophoresis used to separate DNA fragments of different lengths?

Answer 14

Gel electrophoresis uses an electric field to drive DNA molecules through a gel that acts as a molecular sieve. The gel is an aqueous matrix of agarose or polyacrylamide. DNA molecules are loaded into a slot or well at one end of the gel. When an electric field is applied, the negatively charged DNA molecules migrate toward the positive electrode. Shorter DNA molecules are less hindered by the agarose or polyacrylamide matrix and migrate faster than longer DNA molecules, which must wind their way around obstacles and through the pores in the gel matrix.

Question 15

After DNA fragments have been separated by gel electrophoresis, how can they be visualized?

Answer 15

DNA molecules can be visualized by staining with a fluorescent dye, such as ethidium bromide, that intercalates between the stacked bases of the DNA double helix, and the dye–DNA complex fluoresces when irradiated with an ultraviolet light source. Alternatively, they can be visualized by attaching radioactive or chemical labels to the DNA before it is placed in the gel.

Question 16

What can gel electrophoresis not determine?

Answer 16

Nothing about the sequences, but only the size of the fragment

Question 17

What is the purpose of southern blotting?

Answer 17

Southern blotting is used to detect and visualize specific DNA fragments that have a sequence complementary to a labeled DNA probe.

Question 18

How is Southern blotting carried out?

Answer 18

DNA is first cleaved into fragments with restriction endonucleases. The fragments are separated by size via gel electrophoresis. These fragments are then denatured and transferred by blotting onto the surface of a membrane filter. The membrane filter now has single-stranded DNA fragments bound to its surface, separated by size as in the gel. The filter is then incubated with a solution containing a denatured, labeled probe DNA. The probe DNA hybridizes to its complementary DNA on the filter. After washing away excess unbound probes, the labeled probe hybridized to the DNA on the filter can be detected using the appropriate methods to visualize the label. For radioactively labeled probes, the bound probe is detected by exposure to X- ray film. Other probe labeling methods detect bound probe using enzymatic reactions that generate luminescence or color.

Question 19

Give three important characteristics of cloning vectors

Answer 19

(1) An origin of DNA replication so they can be maintained in a cell
(2) A gene, such as antibiotic resistance, to select for cells that carry the vector
(3) A unique restriction site or series of sites to where a foreign DNA molecule may be inserted

Question 20

Briefly describe two different methods for inserting foreign DNA into plasmids

Answer 20

Restriction cloning: Vector and foreign DNA are cut with the same restriction enzyme, then ligated together with DNA ligase.
PCR fragment cloning: DNA fragments generated by PCR may be ligated to plasmid vectors in either of two ways. One way is to synthesize PCR primers that have restriction sites at or near their 5′ ends. The resulting PCR fragments can be digested with the appropriate restriction enzymes to generate sticky ends for restriction cloning as described above.

Question 21

Briefly explain how an antibiotic-resistance gene and the lacZ gene can be used to determine which cells contain a particular plasmid

Answer 21

Many plasmids designed as cloning vectors carry a gene for antibiotic resistance and the lacZ gene. The lacZ gene on the plasmid has been engineered to contain multiple unique restriction sites. Foreign DNAs are inserted into one of the unique restriction sites in the lacZ gene of plasmids and the plasmids are transformed into E. coli cells lacking a functional lacZ gene. Transformed cells are plated on a medium containing the appropriate antibiotic to select for cells that carry the plasmid. The medium also contains an inducer for the lac operon, so the cells express the lacZ gene, and X-gal, a substrate for beta-galactosidase that will turn blue when cleaved by β-galactosidase. The colonies that carry plasmid without foreign DNA inserts will have intact lacZ genes, make functional β-galactosidase, cleave X-gal, and turn blue. Colonies that carry plasmid with foreign DNA inserts will not make functional β-galactosidase because the lacZ gene is disrupted by the foreign DNA insert. They will remain white. Thus, cells carrying plasmids with inserts will form white colonies. This is known as blue/white selection.

Question 22

Briefly explain how the polymerase chain reaction is used to amplify a specific DNA sequence

Answer 22

First, the double-stranded template DNA is denatured by high temperature. Then, primers corresponding to the ends of the DNA sequence to be amplified are annealed to the single-stranded DNA template strands. These primers are extended by a thermostable DNA polymerase so that the target DNA sequence is duplicated. These steps are repeated 30 times or more. Each cycle of denaturation, primer annealing, and extension results in doubling the number of copies of the target sequence between the primers.

Question 23

What are some of the limitations of PCR?

Answer 23

PCR amplification is limited by several factors. One is that sequence of the gene to be amplified must be known, at least at the ends of the region to be amplified, in order to synthesize the PCR primers. Another is that the extreme sensitivity of the technique renders it susceptible to contamination. A third limitation is that the most common thermostable DNA polymerase used for PCR, Taq DNA polymerase, has a relatively high error rate. A fourth limitation is that PCR amplification is usually limited to DNA fragments of up to a few thousand base pairs; optimized DNA polymerase mixtures and reaction conditions extend the amplifiable length to around 20 kb.

Question 24

What is real-time PCR?

Answer 24

Real-time PCR uses fluorescent probes to detect the formation of specific PCR products, and a sensitive instrument to quantify the amount of PCR product formed after each cycle of PCR, while the reaction is proceeding. This technique is used to quantify absolute or relative amounts of template DNA in samples.

Question 25

How does a genomic library differ from a cDNA library?

Answer 25

A genomic library is generated by cloning fragments of chromosomal DNA into a cloning vector. Chromosomal DNA is randomly fragmented by shearing or by partial digestion with a restriction enzyme. A cDNA library is made from mRNA sequences. Cellular mRNAs are isolated and then reverse transcriptase is used to copy the mRNA sequences to cDNA, which are cloned into plasmid or phage vectors. Therefore a cDNA library only represents the genes expressed in the tissues of origin for the RNA.

Question 26

Briefly explain in situ hybridization, giving some applications of this technique

Answer 26

In situ hybridization involves hybridization of radiolabeled or fluorescently labeled DNA or RNA probes to DNA or RNA molecules that are still in the cell. This technique can be used to visualize the expression of specific mRNAs in different cells and tissues and the location of genes on metaphase or polytene chromosomes.

Question 27

What is the function of Taq polymerase in the PCR reaction?

Answer 27

Stable DNA polymerase at high temperature

Question 28

Briefly explain how a gene can be isolated through positional cloning

Answer 28

The approximate location of a gene on a chromosome is identified by recombination or deletion mapping, with markers and deletions with known positions on the chromosome. All genes within this region are characterized to determine which gene has mutations that cosegregate with mutant phenotypes.

Question 29

Explain how chromosome walking can be used to find a gene

Answer 29

Chromosome walking is used to isolate DNA clones encoding genes defined solely by mutations, for which no DNA or amino acid sequence is known. Mapping experiments locate the gene to a chromosome and to a region of the chromosome. The chromosome walk begins with a neighboring gene that has been previously cloned. Clones that overlap this initial gene are isolated, then the overlapping clones are used to isolate additional overlapping clones, until the set of overlapping clones spans the entire chromosomal region that may contain the target gene. All genes identified within this region are characterized to determine which gene contains mutations that cosegregate with the mutant phenotype.

Question 30

What is the purpose of the dideoxynucleoside triphosphate in the dideoxy sequencing reaction?

Answer 30

Dideoxynucleoside triphosphates (ddNTPs) act as a substrate for DNA polymerase but cause termination of DNA synthesis when they are incorporated, because they lack the 3′–OH for the addition of the next nucleotide. Mixed with regular dNTPs, fluorescently labeled ddNTPs generate a series of DNA fragments that have terminated at every nucleotide position along the template DNA molecule being sequenced. These fragments can be separated by gel electrophoresis. Because each of the four ddNTPs carries a different fluorescent label, a laser detector scanning near the end of the gel can distinguish which base terminates each fragment. Reading the fragments from shorter to longer, an automated DNA sequencer can determine the sequence of the template DNA molecule.

Question 31

What is DNA fingerprinting?

Answer 31

DNA fingerprinting is the typing of an individual for genetic markers at highly variable loci. This is useful for forensic investigations, to determine whether the suspect could have contributed to the evidentiary DNA obtained from blood or other bodily fluids found at the scene of a crime. Other applications include paternity testing and the identification of bodily remains.

Question 32

What types of sequences are examined in DNA fingerprinting?

Answer 32

The first loci used for DNA fingerprinting were variable number of tandem repeat (VNTR) loci; these consist of short tandem repeat sequences located in introns or spacer regions between genes. The number of repeat sequences at the locus does not affect the phenotype of the individual in any discernible way, so the numbers of repeats at each of these loci are highly variable in the population. More recently, loci with smaller repeat sequences of just a few nucleotides, called short tandem repeats (STRs), have been adopted because they can be amplified by PCR. The variable number of repeats creates PCR fragments of different sizes. Genotyping at 13 to 15 of these unlinked STR loci can identify one individual among trillions of potential genotypes.

Question 33

Briefly explain how site-directed mutagenesis is carried out

Answer 33

In oligonucleotide-directed mutagenesis, an oligonucleotide containing the desired mutation in the sequence is synthesized. This mutant oligonucleotide is annealed to denatured target DNA template and used to direct DNA synthesis. The result is a double-stranded DNA molecule with a mismatch at the site to be mutated. When transformed into bacterial cells, bacterial repair enzymes will convert the molecule to the mutant form about 50% of the time.

Question 34

What are knockout mice?

Answer 34

Knockout mice have a target gene disrupted or deleted (“knocked out”).

Question 35

How are knockout mice produced?

Answer 35

First, the target gene is cloned. The middle portion of the gene is replaced with a selectable marker, typically the neo gene that confers resistance to the drug G418. This construct is then introduced back into mouse embryonic stem cells and cells with G418 resistance are selected. The surviving cells are screened for cells where the chromosomal copy of the target gene has been replaced with the neo-containing construct by homologous recombination of the flanking sequences. These embryonic stem cells are then injected into mouse blastocyst-stage embryos and these chimeric embryos are transferred to the uterus of a pseudopregnant female mouse. The knockout cells will participate in the formation of many tissues in the mouse fetus, including germ-line cells. The chimeric offspring are interbred to produce offspring that are homozygous for the knockout allele.

Question 36

How are knockout mice useful?

Answer 36

The phenotypes of the knockout mice provide information about the function of the gene.

Question 37

List some of the uses of PCR

Answer 37

Detecting the presence of viruses in blood samples
Identify genetic variation
Isolate DNA from ancient sources
Amplify small amounts of DNA from crime scenes
Introduce new sequences into a fragment of DNA

Question 38

How is RNA interference used in the analysis of gene function?

Answer 38

RNA interference is one potential reverse genetics approach to analyze gene function, by specifically repressing expression of that gene. Double-stranded RNA may be injected directly into a cell or organism or the cell or organism may be genetically modified to express a double-stranded RNA molecule corresponding to the target gene.

Question 39

What is gene therapy?

Answer 39

Gene therapy is the correction of a defective gene by either gene replacement or the addition of a wild-type copy of the gene. For this to work, enough of the cells of the critically affected tissues or
organs must be transformed with the functional copy of the gene to restore normal physiology.

Question 40

What is a DNA library?

Answer 40

A collection of clones containing all the DNA fragments from one source

Question 41

What is a cDNA library?

Answer 41

Consisting only of those DNA sequences that are transcribed into mRNA

Question 42

What is the most common fatal genetic disease in the US today?

Answer 42

Cystic fibrosis

Question 43

What is CF?

Answer 43

autosomal recessive disease characterized by chronic lung infections, insufficient pancreatic enzyme production, and increased salt concentration in sweat
CFTR gene is necessary to maintain fluid balance