Tools of Molecular Bio

Question 1

Recombinant DNA

Answer 1

DNA mlc produced artificially and containing seq. from unrelated organisms.

Question 2

Subcloning

Answer 2

The process of inserting a DNA sequence into a vector

Question 3

Creating recombinant DNA

Answer 3

1. ) Vector (plasmid DNA with drug resistance genes) plus DNA of interest cleaved with restriction endonuclease
2. ) DNA samples mixed, joined by DNA ligase
3. ) Host cells made to take up dNA hybrids, and transformants selected by drug resistance
4. ) Bacteria carrying recombinant plasmids must be probed or screened to id presence of gene of interest

Question 4

Restriction endonucleases

Answer 4

Cut double stranded DNA at specific sequences, restriction sites are palindromes
Produce sticky/cohesive or blunt ends depending on enzyme
Allow specific/reproducible fragmentation of DNA

Question 5

DNA ligase

Answer 5

Joins 5’ phosphate end and 3’ OH end (can be different mlcs)

Question 6

Plasmid

Answer 6

Extrachromosomal DNA in a bacterial cell that replicates independently, but does NOT INTEGRATE INTO HOST chromosome.
Used as a vector for DNA cloning in E. coli

Plasmids used in recombinant DNA technology have been artificially created by recombining fragments of various existing plasmids.
Plasmids contain multiple cloning sites that are sequences recognized by restriction endonucleases.

May contain antibiotic resistance gene so that you can id them on an agar with the antibiotic

Question 7

Transformation

Answer 7

Bacterial cell made competent to take up plasmid DNA

Question 8

How to characterize a piece of DNA?

Answer 8

1. By restriction digestion analysis: (quickest and cheapest method).
2. By hybridization: (used when many clones have to be screened).

3. By DNA Sequencing: (used when trying to identify a mutated recombinant clone).
4. By PCR amplification – The Polymerase Chain Reaction (PCR):

Question 9

Restriction digestion analysis

Answer 9

• Bacteria containing recombinant plasmids are grown as clones.
• DNA is isolated from multiple clones and digested with enzymes that would indicate by the sizes of DNA produced whether the DNA segment of interest had been inserted.

• The pattern of restriction endonuclease digestion fragments as determined by gel electrophoresis and ethidium bromide staining of DNA (quickest and cheapest method).

Question 10

Hybridization

Answer 10

• The clones are blot transferred to a membrane sheet, and the DNA present denatured and fixed onto the surface.

• Adding a radioactive “probe” or complementary fragment and allowing the DNA to hybridize followed by exposure to X-ray film identifies the clone containing recombinant DNA with the correct insert.


Question 11

By DNA sequencing

Answer 11

• The normal substrates for DNA synthesis are nucleoside triphosphates that are based on the sugar 2-deoxyribose (dNTP).
• 
When 2,3-dideoxyribose (ddNTP) is incorporated into the DNA backbone, DNA synthesis is terminated.
• A primer sequence is determined for an unknown fragment of single-stranded DNA. This unknown DNA is combined with DNA polymerase, primer, dNTPs and ddNTPs in four separate reactions containing either a T,C,G or C ddNTP for manual sequencing, or with all four ddNTPs which have each been marked with a different fluorescent dye for automated sequencing.

• If a dNTP is incorporated into the growing strand, DNA synthesis continues. If a ddNTP is used, however, synthesis is terminated.

• DNA synthesis is allowed to continue until many different fragments of varying lengths are produced. These fragments are separated by gel electrophoresis.
• The terminating ddNTP is identified by the reaction (for manual sequencing) or its color (automated sequencing), and the sequence can be “read” from the bottom of the gel to the top.

• The sequence of the newly synthesized DNA (which is deduced from the gel) is the complement of the unknown strand.


Question 12

PCR

Answer 12

• Primers are synthesized that are complementary to both strands of a specific sequence of DNA.
• A thermally stable DNA polymerase (such as Taq) the four nucleotide triphosphates are added to the reaction containing primers and DNA template. DNA strands are denatured by heating to ~95 ̊C and the solution cooled to ~55 ̊C to allow the primers to hybridize to the template.
• The polymerase uses each strand as a template and makes a copy by extending the 3’ end of the primer, at around ~72 ̊C.
Heat and cool, process repeats. Repeat it a shit ton. Get lots of copies of gene!

Question 13

Choosing a vector

Answer 13

Plasmids are used in amplification of DNA sequences in bacteria, and can be introduced into cells of higher eukaryotes (won’t replicate in these organisms)

Bacteriophage: to infect E. coli when high efficiency of intro of recombinant vector is required

Cosmids: hybrids of plasmids and bacteriophage, but unlike bacteriophage they do not kill the host cell

Viral and artifical chromosmal vectors: introduce DNA into eukaryotic tissues/ can carry HUGE DNA inserts

Viral vectors: used when you want integration into the host genome (like gene therapy)

Question 14

Southern blotting

Answer 14

ID DNA frag from complex mix of fragments.
Denatured and seprated by gel electophoresis, blotted onto mem, reacted with radioactive probe, binds complementary DNA seq, autioradiography to detect

Question 15

Norther

Answer 15

similar method to southern, but RNA

Question 16

Primers

Answer 16

single stranded oligonucleotdies of any sequence, can be made.
Oligos or primers are key to DNA sequencing and PCR. Often labaelled and used as probes for hybridization analyses.

Question 17

Hybridization

Answer 17

Core component of many bio techniques. Steps:

1. ) denature DNA so complimentary base pairs are accessible for hybridization to the primer/probe
2. ) Anneal the primer/probe at temp just below melting (essential for preventing non-specific hybridization). Want Tm below Tm of primer/probe
3. ) Add primer or probe in excess so it can out-compete the other template single strand of DNa for binding to the complimentary single strand of DNA

Question 18

Microarrays

Answer 18

ORdered arrays of oligonucleotide primers that hybridize to RNA or DNA targets of interest.
Typically samples are fluorescently labelled, hybridized to microarray, and the level of RNA or DNA in sample is inferred from target intensity

Question 19

Paternity testing

Answer 19

by DNA fingerprinting of variable number tandem repeats. Consists of PCR or restriction digestion followed by southern blot of region with VNTR, then electrophoreses and detection of altered size of DNA frag patterns

Question 20

Can follow inheritance of a disease by….

Answer 20

Linkage to microsatellite repeats or minisatellite repeats, uses PCR amplification via primers flanking the microsatellite repeats

Question 21

Screening for disease allele with hybridization

Answer 21

The allele must have been previously characterized. Can test for presence of multiple disease alleles at the same time. Oligonucleotides are designed which hybridizes to the specific disease alleles but not to the normal allele and these allele-specific oligonucleotides are immobilized to a membrane. The patients DNA in the region(s) spanning the location of the mutant alleles is amplified and labeled by PCR and hybridized to the allele-specific oligonucleotides. Problematic if different disease alleles are very close together – as this will lead to no hybridization and a false negative signal.

Question 22

Screening for disease alleles using allele-specific PCR

Answer 22

The allele(s) must have been previously characterized. Detects small mutations, such as point mutations or small insertions/deletions. The PCR primers are designed so that they will hybridize with the mutant allele but not with the normal allele. The presence of a PCR product indicates the presence of the disease allele. Can test for presence of any one of multiple disease alleles at the same time by designing the reaction such that the PCR product from each mutant allele is a different size. Used for diagnosing 100ʼs of genetic alterations: Cystic fibrosis, Tay-Sachs disease, Beta-thalassaemia, HLA typing.

Question 23

DNA genotyping

Answer 23

uses microarray or similar technology to determine the status of single nucleotide polymorphisms at specific base location in a genome. Particularly useful for determining mutation status (including heterozygosity) as well as haplotype status (e.g. HLA and CYP loci).