Recombinant DNA, Concepts and Applications

Question 1

what is sanger sequencing?

Answer 1

Most common procedure for determining the sequence of nucleotides in a DNA strand by using dideoxynucleotides (ddNTP)

Question 2

what are the steps used in sanger sequencing?

Answer 2

Only 1 of the 4 ddNTPs (ddATP, ddGTP, ddCTP, ddTTP) is added to a tube containing all 4 normal dNTP, DNA polymerase, a primer, and the template strand for the DNA being sequenced (4 separate reactions)

ddNTPs compete with its corresponding normal nucleotide for insertion into the growing chain

When a ddNTP is incorporated, polymerization is terminated

Some of the chains will terminate at each of the locations in the template strand that is complementary to the ddNTP

DNA sequence (5’—>3’) can be determined by “reading” from the bottom to the top of the gel

Question 3

what is PCR and its goal?

Answer 3

a method of amplifying a selected DNA sequence and does not rely on the biologic cloning method.

PCR permits the synthesis of millions of copies of a specific nt sequence in a few hours even when the targeted sequence makes up <1 ppm of the total initial sample.

Question 4

what are the steps in PCR?

Answer 4

denature DNA into separate strands by increasing temperature

anneal primers to flanking regions of single stranded DNA

extend primers with DNA polymerase

the two new double stranded DNA molecules can be denatured and copied by steps 1 to 3

Question 5

what are the PCR requirements?

Answer 5

DNA template - the one interested in amplifying

Oligonucleotide primers - short nucleotide segments that flank the DNA target sequences

dNTPs (dATP, dCTP, dGTP, dTTP)

Heat-stable DNA polymerase to prevent protein denaturation

Question 6

what can be done with the amplified DNA products?

Answer 6

they can be separated by gel electrophoresis, detected by Southern blotting and hybridization, and sequenced

Question 7

what is the advantage of using PCR over cloning?

Answer 7

sensitivity and speed

Question 8

what is the purpose of the southern blot?

Answer 8

detects large changes in DNA; detects point mutations that create or destroy restriction sites

Question 9

what is the sample analyzed in the southern blot?

Answer 9

DNA

Question 10

what is the purpose of the northern blot?

Answer 10

measures mRNA amounts and sizes

Question 11

what is the sample analyzed in the northern blot?

Answer 11

RNA

Question 12

what is the purpose of the western blot?

Answer 12

measures protein amounts

Question 13

what is the sample analyzed in the western blot?

Answer 13

protein

Question 14

in gel electrophoresis, how will the sample travel along the gel after electricity is supplied?

Answer 14

The gel acts as a permeable matrix (a sieve) through which molecules can travel when an electric current is passed across it

B/C DNA is negatively charged at neutral pH, it will migrate towards the positive electrode

Shorter fragments migrate more rapidly

Bands of DNA can be visualized by staining with dyes (e.g. EtBr) or a labeled probe

Question 15

what is recombinant DNA?

Answer 15

Molecules of DNA from different sources that have been recombined in vitro (outside the organism)

Question 16

briefly describe the steps used in producing recombinant DNA

Answer 16

restriction endonuclease (must be the same) used to bind DNA back together using DNA ligase used and so you have what is called recombinant DNA or chimeric DNA

*Sticky ends of 2 unrelated DNA fragments can be joined together if ends are complementary (cut w/ same restriction enzyme)

Question 17

what is DNA cloning? what do you need for this?

Answer 17

the introduction of a foreign DNA molecule into a replicating cell for amplification;

DNA of interest and vector (carrier DNA of the molecule of interest into another cell) to make copies

Question 18

with respect to cloning, what is bacteria host?

Answer 18

(“transformed cells”)

Bacteriophage ,plasmids, cosmids

Question 19

with respect to cloning, what is eukaryotic host?

Answer 19

(“transfected cells”)

Retroviruses, adenoviruses, free DNA, or DNA coated with a lipid layer (liposome)

Question 20

What is the value of fluorescent tags attached to molecules?

Answer 20

Fluorescent probes can be used to localize specific sequences within chromosomes

Question 21

what information can be provided through use of the fluorescent tags?

Answer 21

Labelled probes can allow for detection and localization of DNA or RNA sequences in cell or tissue preparations in a process called in situ hybridization

If the probe is fluorescent, the technique is called fluorescent in situ hybridization or FISH and can be used to check for chromosomal additions or deletions. Fish can also be used to probe chromosomes with different colors and look for conditions like anaploidy (change in some chromosomes) and polyploidy (change in a whole set of chromosomes)

Question 22

what are DNA polymorphisms?

Answer 22

a change in genotype that can result in no change in phenotype or a change in phenotype is harmless; causes increased susceptibility of disease; or, rarely causes the disease.

Traditional definition: A sequence variation at a given locus (allele) in more than 1% of a population, this is how it is different than a mutation

Question 23

what are the common forms of polymorphisms?

*consider these as effects on proteins and/or cells

Answer 23

RFLP: restriction fragment length polymorphism

VNTR: variable number tandem repeats

STR: short tandem repeats (1-8bp)

SNP: single-nucleotide polymorphisms

Question 24

with relation to DNA polymorphisms, what would we consider Sickle cell anemia?

Answer 24

an RFLP which is a genetic variant that can be observed by cleaving the DNA into fragments (restriction fragments) with a restriction enzyme

Question 25

what else can we use to detect sickle cell anemia?

Answer 25

Allele-specific Oligonucleotide Probes (ASOs)

Question 26

how can we use ASOs to detect sickle cell anemia?

Answer 26

IF the sequence of all or part of the target DNA is known….

ssDNA oligonucleotide probes can be synthesized that are complementary to a small region of the gene of interest (allele-specific)

Oligonucleotide probes can be used to detect single-base changes in the sequence to which they are complementary.

The genomic region that contains the abnormal gene is amplified by PCR, and the samples are “dotted” on nitrocellulose paper.

The paper is treated with a labelled probe for either the normal or mutant sequence

Detection will indicate whether the normal or mutant probe has preferentially hybridized with the DNA

Carriers will have two different alleles – one that binds to the normal and mutant probe

*helps in determination of sickle cell anemia of specific allele

Question 27

how is VNTR used in the clinical setting?

Answer 27

Human DNA contains many sequences that are repeated in tandem a variable number of times at certain loci in the genome.

These regions are called highly variable b/c they contain a variable # of tandem repeats (VNTR)

Digestion with restriction enzymes that recognize sites that flank the VNTR region produces fragments containing these loci

Fragments differ in size from one individual to another, depending on the # of repeats present

use of southern blotting can cut out restriction sites for specific allele run on gel to separate out used on size and then probe VNTR site with DNA sequence and is called DNA fingerprinting and forensic

Question 28

what type of DNA polymorphism can we use to identify Huntington’s Disease?

Answer 28

Huntington’s disease (HD) is anautosomal dominant progressive neurodegenerative disorder. It can be identified at a VNTR on an allele at the HTT gene.

Question 29

what is used when look to compare differences in a normal gene and mutant gene? Low- abundance of nucleic acid sequence? DNA samples?

Answer 29

PCR

Question 30

what are DNA chips used for?

Answer 30

Application which permits screening of many genes simultaneously to determine which alleles of these genes are present in samples obtained from patients

Question 31

since we know that Comparative Genomic Variation is a type of Microarray, how can we use it clinically?

Answer 31

Comparative genomic hybridization (CGH) provides an alternative means of genome-wide screening for copy number variations

CGH enables the identification of genomic regions that may be increased or decreased in copy number. Copy number aberrations are common tumor determinates. Increased copy numbers of genes whose products push the cell cycle forward may result increased cell division. Decreased copy numbers of genes whose products are involved in checkpoints to prevent unnecessary cell division can result in uncontrolled cell division.

Question 32

what else can microarrays be used for?

Answer 32

Determine which one of the many known mutations of a particular genetic disease is the specific defect underlying a patients problem

Determine which alleles of drug-metabolizing enzymes are present, and therefore, the likelihood of that individual having an adverse reaction to a particular drug

Diagnosis of infectious disease

Gene expression levels in various diseased states vs. normal

Question 33

how are SiRNA’s used in therapeutic applications?

Answer 33

SiRNA (Small interfering RNAs) are chemically synthesized RNA molecules which neutralize/inhibit targeted mRNA molecules.

SiRNAs utilize the process and machinery
of microRNAs (miRNAs) to “knock down” mRNA  expression and translation.

Promising for cancer therapeutics