tools of molecular biology.2

Question 1

Recombinant DNA

Answer 1

are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms.

Question 2

Genetic Engineering

Answer 2

Use of techniques involving recombinant DNA technology to produce molecules and/or organisms with new properties.

Question 3

Subcloning

Answer 3

The process of inserting a DNA sequence into a vector.

Question 4

Biotechnology

Answer 4

All-inclusive term for several technologies including but not limited to recombinant DNA. Refers to the use of technology in applications for solving fundamental problems in biology.

Question 5

Creating Recombinant DNA

Answer 5

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

Question 6

reverse transcriptase

Answer 6

an enzyme used to generate complementary DNA (cDNA) from an RNA template. The cDNA encoding a human protein, in this case insulin is inserted into a bacterial plasmid by the techniques we talked about earlier, It is important that you realize that it is the cDNA that has to be inserted i.e DNA without the introns, so you need to know how you make a cDNA. A good place to put your primer is the poly A tail

Question 7

Restriction endonucleases

Answer 7

also called restriction enzymes, cut double stranded DNA at specific sequences, - restriction sites, which are usually palindromes: a sequence which is the same when read from either DNA strand in the 5ʼ-3ʼ direction. i.e. TACGTA. Restriction enzymes produce either sticky/cohesive (overhangs) or blunt ends, depending on the particular enzyme used. Restriction endonucleases allow the specific and reproducible fragmentation of DNA. The discovery of these enzymes allowed the development of modern recombinant DNA technology. 


Question 8

CRISPR-mediated interference

Answer 8

are DNA loci containing short repetitions of base sequences. Each repetition is followed by short segments of “spacer DNA” from previous exposures to a virus. the CRISPR/Cas system has been used for gene editing (adding, disrupting or changing the sequence of specific genes) and gene regulation in species throughout the tree of life.[7] By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism’s genome can be cut at any desired location.

Question 9

Cas9 (CRISPR associated protein 9)

Answer 9

an RNA-guided DNA nuclease enzyme associated with Streptococcus pyogenes’ CRISPR immunity system. Cas9 is widely used to induce site-directed double strand breaks in DNA, which can lead to gene inactivation or the introduction of heterologous genes through non-homologous end joining and homologous recombination respectively. Because it can cleave almost any sequence if supplied with a guide RNA containing that same sequence, Cas9 is much easier to use than earlier methods that required new proteins to constructed for each site to be cut. This flexibility also allows Cas9 to turn genes on and off by localizing transcriptional activator or repressors to specific DNA sequences.

Question 10

DNA ligase

Answer 10

joins 5’-phosphate and 3’-hydroxyl ends of DNA to form a phosphodiester link. Two fragments formed by a restriction enzyme such as EcoRI can be rejoined by ligase. Similarly, EcoRI fragments from two different pieces of DNA can be joined. The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3’ hydroxyl ends of one nucleotide, (“acceptor”) with the 5’ phosphate end of another (“donor”). ATP is required for the ligase reaction, which proceeds in three steps: 1) adenylation (addition of AMP) of a lysine residue in the active center of the enzyme, pyrophosphate is released; 2) transfer of the AMP to the 5’ phosphate of the so-called donor, formation of a pyrophosphate bond; 3) formation of a phosphodiester bond between the 5’ phosphate of the donor and the 3’ hydroxyl of the acceptor.

Question 11

essential features to a cloning vector

Answer 11

1 need Restriction sites for ligating in new pieces of DNA that we are studying.
2. In order for copies of the plasmid to be made, or ‘cloned’ the plasmid also needs an origin of replication. When introduced into a cell, this sequence allows the plasmid to be recognized by host factors that replicate the plasmid and segregate it to a daughter cell during cell division.
The third feature that you need to know about that a plasmid must have is a means to select for the plasmid. Usually this is in the form of an antibiotic resistance gene, such as ampicillin resistance.
By growing bacteria on a growth media containing an antibiotic, only those bacteria that contain the plasmid will be resistant to the antibiotic and will grow.

Question 12

Plasmid

Answer 12

Extrachromosomal DNA in a bacterial cell which can replicate independently but which cannot integrate into the host chromosome. Used as a vector for DNA cloning in the bacteria E. coli. Drug resistance plasmids are not essential for the cell’s growth, but confer antibiotic resistance. Plasmids that are used for 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.


Question 13

Transformation

Answer 13

bacterial cell made competent to take up plasmid DNA.

Question 14

Antibiotic resistance

Answer 14

Plasmid vector contains an antibiotic resistance gene making the cell resistant. Growth of transformed cells (cells receiving the plasmid) can be identified on agar medium containing the antibiotic.

Question 15

Ways to characterize DNA

Answer 15

1. By restriction digestion analysis, 2. By hybridization, 3. By DNA Sequencing, and By PCR amplification

Question 16

restriction digestion analysis

Answer 16

This is the quickest and cheapest method. 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 17

hybridization DNA

Answer 17

This is used when many clones have to be screened. The two strands of a DNA molecule are denatured by heating to about 100°C = 212°F (a to b). At this temperature, the complementary base pairs that hold the double helix strands together are disrupted and the helix rapidly dissociates into two single strands. Similar hybridization reactions can occur between any single stranded nucleic acid chain: DNA/DNA, RNA/RNA, DNA/RNA. If an RNA transcript is introduced during the renaturation process, the RNA competes with the coding DNA strand and forms double-stranded DNA/RNA hybrid molecule (c to d). These hybridization reactions can be used to detect and characterize nucleotide sequences using a particular nucleotide sequence as a probe. A probe is a labeled piece of DNA. is a core component of many molecular biology techniques, including sequencing, southern and Northern blotting and, PCR.

Question 18

Hybridization with blotting techniques

Answer 18

With blotting techniques, 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 19

DNA Sequencing

Answer 19

used when trying to identify a mutated recombinant clone. 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 20

By PCR amplification

Answer 20

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. If you heat again and then cool, the process repeats. Repeating this cycle 35 times gives 235 or 10 billion copies of the gene. 


Question 21

Choice of vectors for DNA cloning

Answer 21

Plasmids are the vector of choice for amplification of DNA sequences in bacteria, and plasmids can also be introduced into cells of higher eukaryotes (but will not replicate in these organisms). Bacteriophage are used to infect E. coli when high efficiency of introduction of the recombinant vector is required (take up to a 20kb insert). Cosmids are hybrid of plasmids and bacteriophage – unlike bacteriophage they do not kill their host cell, they use a plasmid replication origin and can carry up to 45kb of foreign DNA. Viral and artificial chromosomal vectors allow introduction of DNA into eukaryotic tissues and / or can carry huge DNA inserts. Viral vectors, such as retroviruses, are used where integration into the host genome is required such as during gene therapy.

Question 22

cosmid

Answer 22

If your piece of DNA of interest is larger than 25kb, then you can use cosmids are plasmids that have sequences that allow the cosmid DNA to be packaged into lambda bacteriophage. The cosmid encodes no viral products therefore forms no plaques, but can select for antibiotic resistance of the plasmids. Since many human genes are up to 45kb longer, cosmid cloning increases the chance of obtaining DNA clones containing the entire sequences of genes.

Question 23

BAC’s

Answer 23

a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome’s usual insert size is 150-350 kbp, which are larger than other plasmids

Question 24

artificial chromosomes

Answer 24

are large pieces of DNA that resemble chomosomes.

Question 25

retroviruses in gene therapy

Answer 25

If you want to use your vector to deliver a DNA sequence into a mammalian cell, then the vector needs to be able to replicate in human cells. And sometimes it is desirable to introduce these pieces of DNA into the human genome. For this case, viruses are often used, particularly retroviruses. These are often used to deliver genes into organisms for gene therapy.

Question 26

Yacs

Answer 26

are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae, which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from 100–1000 kb, the inserted sequences can be cloned and physically mapped using a process called chromosome walking. They have centromere

Question 27

transformation

Answer 27

The process of introducing plasmids into DNA. This is inefficient, therefor you need a selection marker

Question 28

some recombinant DNA used in clinic

Answer 28

In the clinic, you will be using dozens of different recombinant proteins including insulin, growth factors, blood factors, vaccines that were produced from recombinant DNA molecules to treat genetic diseases which result in insufficient clotting factors such as Classic Hemophilia (Factor VIII-Deficiency). Recombinant human proteins are also be used to augment normal processes such as wound healing and immune system function.

Question 29

Gel electrophoresis

Answer 29

Used to separate DNA molecules on the basis of their size. DNA has a negative charge, and in an electric field migrates towards a positive electrode. The rate of migration through a gel is inversely proportional to size. So in order to visualize separated DNA bands on a gel, the gel is soaked in a solution of the fluorescent dye ethidium bromide. This is a planar molecule that intercalates between the base pairs. Binding to DNA increases the intrinsic fluorescence of ethidium. As a result, when the gel is illuminated with UV light, the regions of the gel containing DNA fluoresce much more brightly than the regions of the gel without DNA.

Question 30

Tm

Answer 30

Tm=2˚C(A+T) + 4˚C(G+C), the point at which 50% of oligonucleotide is hybridized to its perfect DNA complement

Question 31

Southern blotting

Answer 31

Used to identify DNA fragment of known sequence from a complex mixture of DNA fragments. DNA fragments denatured and separated by gel electrophoresis. Fragments are blotted onto a sheet of nitrocellulose membrane. Blot is reacted with a radioactive DNA probe which binds to the complementary DNA sequence. Autoradiography used to detect radioactive fragments. 


Question 32

Northern blotting

Answer 32

Used to identify RNA fragments of known sequence form a complex mixture of DNA fragments, using a method very similar to southern blotting.

Question 33

Primers

Answer 33

Single stranded oligonucleotides of any desired sequence (up to 100 nucleotides) can be made via automated chemical synthesis. Such “oligos” or “primers” are key to the techniques of DNA sequencing, and PCR. Oligonucleotides are often labeled and used as probes for hybridization analyses.

Question 34

steps for hybridization

Answer 34

1) Denaturing the double- stranded DNA so that the complimentary base pairs are accessible for hybridization to the primer/ or probe. 2) Annealing the primer / probe to the template at a temperature just below the Tm (melting temperature) of the primer or probe – the Tm is determined by a combination of the sequence and length of the primer / probe. This is essential for specific hybridization and preventing non-specific hybridization. 3) Addition of the primer or probe in excess, so that it can out-compete the other template single strand of DNA for binding to the complimentary single strand of DNA.

Question 35

Microarrays

Answer 35

are ordered arrays of oligonucleotide primers that hybridize to RNA or DNA targets of interest. Typically samples are fluorescently labeled, hybridized to the microarray, and the level of RNA or DNA in the sample is inferred from the signal intensity. Developed to simultaneously analyze the expression patterns of thousands of genes. Microarray chips contain hybridization probes complementary to fluorescently-labeled nucleic acids from source of interest. Comparisons in global gene expression can be made between different conditions. Allows you to compare tumor vs. normal, before and after growth factor addition, etc.

Question 36

Microarray data analysis

Answer 36

Computational clustering algorithms applied to many individual microarray experiments. Identify similar patterns of gene expression across gene (rows) or microarray experiments (e.g. cell types) columns

Question 37

Production of recombinant proteins

Answer 37

the introduction of a human (or other) cDNA into a bacterial expression vector enables production of large amounts of medically important recombinant proteins in E. coli – including insulin, human growth hormone, vaccines, blood factors, anticoagulants, interferons, interleukins etc.

Question 38

Paternity testing

Answer 38

by DNA fingerprinting of variable number tandem repeats. Consists of PCR or restriction digestion (followed by southern blotting) of region containing VNTRs, then electrophoresis and detection of altered size of DNA fragment patterns.

Question 39

Screening for disease alleles characterized by restriction fragment length polymorphisms (RFLP)

Answer 39

Disease allele has to lead to a loss or gain of a cleavage site for a restriction endonuclease. The classic example is screening for the HbS mutation of sickle cell anemia that destroys a restriction site for the restriction endonuclease MstII. Digest patients DNA with “diagnostic” restriction enzyme first, followed by southern or PCR analysis, then electrophoresis and detection of altered size of restriction fragment.

Question 40

Screening for disease allele by linkage to microsatellite repeats or minisatellite repeats

Answer 40

Following inheritance of a disease allele by linkage to microsatellite repeats or minisatellite repeats, uses PCR amplification via primers flanking the microsatellite repeats.

Question 41

Screening for disease by allele-specific hybridization

Answer 41

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 42

Screening for disease alleles using allele-specific PCR

Answer 42

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 43

DNA genotyping

Answer 43

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).

Question 44

Sanger sequencings

Answer 44

The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleosidetriphosphates (dNTPs), and modified di-deoxynucleotidetriphosphates (ddNTPs). The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP), while three other nucleotides are ordinary ones. Putting it in a more sensible order, four separate reactions are needed in this process to test all four ddNTPs. Following rounds of template DNA extension from the bound primer, the resulting DNA fragments are heat denatured and separated by size using gel electrophoresis.

Question 45

di-deoxynucleotidetriphosphates (ddNTPs)

Answer 45

terminate DNA strand elongation. These chain-terminating nucleotides lack a 3’-OH group required for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease extension of DNA when a modified ddNTP is incorporated. The ddNTPs may be radioactively or fluorescently labeled for detection in automated sequencing machines.

Question 46

PCR

Answer 46

two oligonucleotides are designed that are complimentary to sequences at either end of the stretch of DNA to be amplified. Therefore, for PCR you have to know the sequences on either side of your piece of DNA.The DNA template is heat denatured into single strands at around 94˚C, then two primers are added in great excess to the denatured DNA, and the temperature is lowered to 50-60˚C. The specific oligos or primers, which are at a very high concentration, hybridize with their complementary DNA sequences in the template DNA. The hybridized oligos then serve as primers for DNA chain synthesis or elongation. The PCR reaction also contains dNTPs and a temperature stable DNA polymerase such as that from Thermus aquaticus (a bacterium that lives in hot springs). This enzyme called Taq, can extend the primerswhen the temperature is raised to around 72˚C, to make a complimentary second strand to the template. The cycle is repeated, the temperature is raised to around 95˚C to denature or separate the DNA strands, then the temperature is lowered to 55˚C to anneal the primers, and then the temperature is raised to 72˚C to allow the polymerase to extend the primer to make the complementary strand - elongation. And repeat for about 25 cycles

Question 47

restriction fragment length polymorphism (RFLP)

Answer 47

a technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites, and to a related laboratory technique by which these segments can be illustrated. In RFLP analysis, the DNA sample is broken into pieces (digested) by restriction enzymes and the resulting restriction fragments are separated according to their lengths by gel electrophoresis.

Question 48

Testing for sickle cell allele

Answer 48

To test whether the patient has the sickle cell allele of the beta globin locus, digest their genomic DNA with MstII, run out the DNA on an electrophoresis gel, and probe with a region spanning the MstII site by southern blotting. What we look for is a longer DNA fragment from the sickle cell allele, as compared to the 2 fragments that we get from the normal allele. But of course we each have two copies of each gene. Sickle cell anaemia is a recessive trait, meaning you only get the disease if you have two sickle alleles, but often you want to know if you are a carrier of the disease, I.e. have one disease allele. So we can use this same analysis to determine who is a carrier or who will have a disease before onset of the symptoms.

Question 49

Screening for cystic fibrosis

Answer 49

1:20 europeans are carriers of this recessive allele. The most frequently occurring mutation, is a deletion of phenylalanine 508 delta-F508. Although the mutant protein is produced since it is only missing one amino acid, it seems to be identified as non-functional and does not undergo the usual final modifications within the endoplasmic reticulum. Instead, the mutant protein is degraded. Primers are designed such that the nucleotide(s) distinguishing the mutant and wild type alleles is at the 3’ terminus of the primer.

Question 50

How do you design PCR primers for dignostic purposes?

Answer 50

If the exact sequence of a disease allele is know, PCR is often used to detect the presence of the disease allele. In this case the mutations are usually small - point mutations, small insertions/deletions. PCR primers corresponding to particular mutations are used in a PCR reaction and the presence or absence of PCR product of expected size indicates the presence or absence of those mutant or normal alleles.

Question 51

Detection of Trinucleotide Repeat Expansion

Answer 51

Triplet repeats are found in a number of human disorders such as Huntington disease (HD), Myotonic dystrophy, spinocerebellar ataxia 1 (SCA 1), Machado-Joseph disease (MJD/SCA 3), Kennedy disease or spinal and bulbar muscular atrophy (SBMA), and dentatorubraopallidoluysian atrophy (DRPLA). These five adult onset neurodegenerative disorders are associated with expansion of a CAG repeat is associated with each of these disorders. A different gene is involved in each disorder. First you can PCR amplification of triplet repeat expansion. Than, the size of the repeat is apparent after gel analysis.

Question 52

DNA fingerprinting

Answer 52

The number of repeats in different individuals varies so much in regions of variable number tandem repeats at different regions of the genome is so great that each single person can be distinguished by a DNA fingerprint based on these sequence variations. This is called DNA fingerprinting and is far superior to conventional fingerprinting - (DUE TO unequal crossing over during meiosis,or Increase or decrease in number of repeats is due to slippage during DNA replication?). The technique of DNA fingerprinting uses either restriction enzymes or PCR to isolate the regions containing the VNTR, then using a southern blot probe for the repeat sequences within the VNTR. Get a set of bands that are unique for each person, and are used extensively in forensic medicine and in determining paternity. DNA fingerprinting takes advantage of the variable number tandem repeats, also called microsatelite DNA. RFLP, so either using Restriction enzymes or PCR products spanning multiple regions of variable nucleotide tandem repeat of a sample from the crime scene get this pattern of bands in an electrophoresis gel