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Module 6: Genetics, evolution and ecosystems > Chapter 21: Manipulating genomes > Flashcards

Chapter 21: Manipulating genomes Flashcards

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Biology 101 flashcards
1
Q

What is agarose gel?

A

Gel used in electrophoresis for separating nucleic acids. It is made of agarose sugar.

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2
Q

What is annealing?

A

Where two complimentary DNA strands join through the formation of H bonds.

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3
Q

What is bioinformatics?

A

The development of software and computer tools needed to store analyse and organise large quantities of raw biological data.

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4
Q

What is computational biology?

A

The study of biology using computational techniques to analyse large amounts of data.

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5
Q

What is ddNTP?

A

A modified DNA nucleotide used in science sequencing that has no 2’ or 3’ of each group. Once incorporated in a DNA strand, no more nucleotides can be added.

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6
Q

What is southern blotting?

A

A technique used to identify specific DNA sequences in fragments separated by electrophoresis.

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7
Q

What is a sticky end?

A

Exposed unpaired nucleotide bases on the ends of cut DNA that have been cleaved by a staggered cut by restriction enzyme.

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8
Q

What is synthetic biology?

A

The design and construction of novel biological pathways, organisms or devices, or the redesign of existing natural biological systems.

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9
Q

What is a vector?

A

A means of inserting DNA from one organism into the cells of another organism. E.g. plasmids, viruses.

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10
Q

What is DNA ligase?

A

An enzyme that catalyse is the formation of phosphodiester bonds between the sugar and phosphate groups of adjacent DNA nucleotides.

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11
Q

What is a DNA probe?

A

Short DNA or RNA sequences complimentary to an own DNA sequence. The probe may be labelled with a radioactive marker or a fluorescent marker.

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12
Q

What is DNA profiling/fingerprinting?

A

Producing an image of the patterns in the non-coding DNA of an individual.

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13
Q

What is DNA sequencing?

A

Working out the sequence of bases in a strand of DNA.

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14
Q

What is Electrophoresis?

A

A type of chromatography that relies on the way charge particles me through a gel under the influence of an electric current. It is used to separate nucleic acid fragments or proteins by sizes.

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15
Q

What is electrofusion?

A

Application of an electric field to recipient cell to make it more receptive to the vector carrying a novel gene.

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16
Q

What is electroporation?

A

The use of a very tiny electric current to transfer genetically engineered plasmids into bacteria or to get DNA fragments directly into eukaryotic cells.

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17
Q

What is germline gene therapy?

A

Inserting a healthy allele into the germ cells (gametes) or into a very early embryo.

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18
Q

What is a Microarray?

A

A high-throughput method of using surface with fixed DNA probes to detect specific DNA sequences in the DNA being studied.

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19
Q

What is high-throughput sequencing?

A

New methods of sequencing that are automated, very rapid and much cheaper than original techniques.

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20
Q

What is a microsatelite/STR?

A

A small non-coding regions of DNA containing a sequence of 2-4 bases repeated 5-15 times. Also known as a short tandem repeat (STR). Used in DNA profiling as each individual has the same STRs at the same loci but with a different number of repeats.

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21
Q

What is a minisatelite/VNTR?

A

A non-coding regions of DNA containing a sequence of 20-50 base pairs repeated many times. Also known as a variable number tandem repeat (VMTR).

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22
Q

What is a plasmid?

A

A small loop of DNA found in bacterial cells. Often used as a vector in genetic modification.

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23
Q

What is a polymerase chain reaction/PCR?

A

A process by which a small sample of DNA can be amplified using specific enzymes and temperature changes.

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24
Q

What is a primer?

A

Short (10-20bp) single-stranded sequences of DNA needed sequencing reactions and PCR.

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25
Q

What is recombinant DNA?

A

New combination of alleles/DNA from two sources.

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26
Q

What is replica plating?

A

A technique used in genetic engineering to identify colonies of transformed bacteria on a petri dish using a selective medium.

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27
Q

What is a restriction enzyme?

A

Endonucleases enzymes that cleave DNA at specific recognition sites.

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28
Q

Describe the structure of DNA.

A

It is made up of two strands coiled to form a double helix. Each strand is made up of nucleotides bound together by phosphodiester bonds. Each nucleotide features of phosphate group, a deoxyribose sugar and a nitrogenous base. The two strands are held together by hydrogen bonds between complimentary base pairs

29
Q

Describe the steps involved in DNA replication.

A

DNA gyrase unwinds the DNA. DNA helicase unzips the DNA creating a replication fork. Both strands act as a template. Free nucleotides pair with the complimentary exposed nucleotides and hydrogen bonds reform. The enzymes DNA polymerase catalyses the joining together of nucleotides synthesising in a 5’ to 3’ direction. Leading strand is synthesised continuously whilst the lagging strand is synthesised as Okazaki fragments. DNA ligase catalyses the joining of Okazaki fragments.

30
Q

Describe the steps involved in transcription.

A

RNA polymerase binds to the promoter region at the start of a gene. The two DNA strands unzip, the hydrogen bonds between complimentary base pairs are broken forming a transcription bubble. One of the DNA strand acts as a template. Free RNA nucleotides from temporary hydrogen bonds with their complimentary base. RNA polymerase joins the RNA nucleotides together in a 5’ to 3’ direction, by catalysing the formation of phosphodiester bonds. Once complete, the pre-mRNA strand breaks off from the template DNA and undergo splicing where the introns are removed before leaving the nucleus.

31
Q

Instead of using PCR, how could we use E. coli to clone DNA.

A

Isolate the DNA using restriction enzymes, place in plasmid of bacteria and provide conditions for bacterial cell to replicate

32
Q

Describe the steps of PCR.

A

Step one is to add the ingredients. These include the DNA template, DNA nucleotides, DNA primers, TAQ polymerase. Step two is heating to 95°C this is denaturation. Hydrogen bonds are broken between complimentary base pairs and the DNA is split into a separate strands. Stage three is cooling to 68°C. This is in Ealing. Specially designed prime is from hydrogen bonds to one end of each single strand of DNA. Stage four is heating to 72° C this is extension. The temperature is increased to keep the DNA strand separated TAQ polymerase binds to primers and catalyses the addition of DNA nucleotides.

33
Q

Why is TAQ polymerase used in PCR?

A

Thermos aquaticus is a bacteria adapted to living in hot Springs. Its enzymes do not denature at high temperatures it is thermostable so we can use it in PCR.

34
Q

Oh is PCR different to natural DNA replication?

A

Only short sequences of up to 10,000 base pairs can be replicated in PCR not entire chromosomes. It requires a series of heating and cooling phases.

35
Q

What are the components involved in electrophoresis?

A

DNA samples, the larger sample that contains DNA fragments, and agarose gel plate, a comb to create wells, and electrophoresis tank which includes the cathode and the anode, a buffer solution which maintains pH and conducts charge, DNA loading die to increase the density of the DNA sample, and DNA binding die which allows visualisation of DNA bonds.

36
Q

Outline the process of gel electrophoresis.

A

Restriction endonucleases are used to cut the DNA samples at specific region site at 34 to 40°C for an hour. Anger is Joe is set up in the tank with a buffer. A comb is removed so well is a left at one end. I don’t loading data is used to help add the digester DNA to the world. Be careful not to pass the bottom of the well using your paper. The DNA samples are put into place and the gel is left. DNA fragments me through the gel the smaller ones travel faster towards the anode. A buffer is poured away and the diet is added to stain the fragments for analysis.

37
Q

Butler in the DNA profiling procedure.

A

Extracting DNA, digesting the sample, separating the DNA fragments, hybridisation and then seeing the evidence.

38
Q

What is the restriction site?

A

Set sequence of bases that fit in the active site of the enzyme.

39
Q

What does PCR do?

A

It amplifies a small section of DNA so copies can be made.

40
Q

Why would a sample with contaminant DNA be a problem for PCR?

A

The contaminant could be amplified as well.

41
Q

What is the enzyme used in the PCR process?

A

TAQ polymerase.

42
Q

What is the reaction mixture in Sanger sequencing?

A

DNA to be sequence, Primus, DNA TAQ polymerase, and activated free nucleotides.

43
Q

What is the difference between normal and modified nuclear tides and explain the effect of these have.

A

Chain terminating is tags with P 32 and has one less oxygen.

44
Q

Outline the steps in the chain termination method.

A

The reaction mixture is added to the PCR machine. It is first heated to 95° to the nature of the DNA which separates the strands. The temperature is lower to around 68° and the primer and News to the template strand DNA TAQ polymerase binds. One type of modified nucleotide DDNTP are then added to each tube. Complimentary faces hydrogen bond together as normal. TAQ polymerase catalyses the addition of new bases in a 5’ to 3’ direction. If a modified nuclear tide is added the polymerase is released and the reaction stops. This continues with many DNA molecules being made.

45
Q

How can we work out the sequence from our electrophoresis gel?

A

Smallest will be at the bottom and the longest will be at the top so read the secrets from the bottom to the top

46
Q

How has this been sped up and automated?

A

Using fluorescent dyes and capillary sequencing. Fragments passed through the capillary tubes towards the anode in order of size. A laser is used to detect a terminating fluorescent base of each sequence.

47
Q

Outline the steps of Pyrosequencing.

A

Step one DNA molecules are broken into fragments. The DNA is then denatured the single-stranded DNA template is used in sequencing. Step three template DNA strands are mobilised. Step for PCR reaction is used to create multiple identical copies per bead. Step 5 1 bead is added per well. Step six the reaction mixture is added. Step seven add one type of activated nucleotide with three phosphate groups. Step eight if a is the next available space on the template strand TPP will be incorporated. Pyrophosphate is hydrolysed into PPI ATP sulfyralayse in the presence of APS converted PPI into ATP. Luciferase converts ATP into Oxy Lucifer in which emits light. That 13 the light is recorded any unincorporated TTP is either greeted by appease. The reaction starts again.

48
Q

ATP is an activated nucleotide and is used ubiquitously in biology as the energy currency of cells. Recently it is also been used in Pyrosequencing. Explain the role of ATP and generating light in Pyrosequencing.

A

PPI released when an activate a nucleotide has been incorporated. In the presence of APS ATP Sophia lease will generate ATP. ATP will allow the conversion of Luciferin into oxyluciferin using luciferase.Oxyluciferin gives off light which is detected by Computer.

49
Q

What are the four ways of isolating genes in the first step of genetic engineering?

A
  1. Using mRNA and reverse transcriptase from retroviruses to create single-stranded cDNA (complementary DNA). DNA polymerase (and primers) synthesise new strand to make it double stranded. 2. Using radioactive or fluorescent probes to locate gene in chino then cut out using restriction enzymes. (sticky ends created with staggered cut). 3. Use an automated polynucleotide synthesiser if you know the gene sequence. 4. Who is PCR to amplify the region from genomic DNA. Need to know gene sequence as need to design appropriate primers.
50
Q

What are the three ways of getting a gene into a vector in the second step of genetic engineering?

A
  1. Plasmids, circular pieces of DNA found in some prokaryotic cells e.g. bacteria such as E. coli. 2. Bacteriophages, viruses that infect bacteria. 3. Attenuated viruses, weakened viruses.
51
Q

How can ligation be used to get a gene into a vector in genetic engineering?

A

Stick/and Elljean into the plasmid. Want the complimentary bases to form H bonds between them. Controlled by enzyme ligase. Like he’s is used to form the phosphodiester bond in the sugar phosphate backbone. Now recombinant DNA.

52
Q

What are the five indirect and direct ways to get a vector into a cell in step 3 of genetic engineering?

A
  1. Electroporation (high voltage pulse). 2. Electrofusion (electrical fields) 3. Transfection (use a bacteriophage) 4. Heat shock treatment (alternate periods of hot- 42°C and cold- 0°C) 4. Ti plasmid and agrobacterium (in plants) direct methods- Gene gun (shoots gold or tungsten covered with DNA into plant cells)
53
Q

What are the stages of genetic engineering for the insulin case study?

A

Step one, obtain desired gene. Adding reverse transcriptase enzyme makes a single strand of sea DNA and treatment with DNA polymerase make a double strand- the gene. Step two, getting gene into vector. Addition of free unpaired nucleotides at the end of DNA produces sticky ends. Step three, get back to into the cell. Now, with the help of like enzymes, the insulin gene can be inserted into plasmids extracted from E. coli bacteria. Now called recombinant plasmid. Step four, recipient cell expresses Jean. E. coli bacteria are mixed with recombinant plasmid and subjected to heat shock in the presence of calcium chloride ions, so this will take up plasmids.

54
Q

Why do scientists extract mature mRNA rather than the gene in genetic engineering?

A

The genomic DNA contains both exons and introns. The mature mRNA has had its introns removed by splicing and only contains exons.

55
Q

What is germline gene therapy?

A

The gene therapy by inserting functional alleles into gametes or zygotes.

56
Q

What is somatic cell gene therapy?

A

The gene therapy by inserting functional alleles into body cells.

57
Q

What causes the genetic disease cystic fibrosis?

A

A lack of a functional CFTR gene. This leads to incorrect chloride channel proteins to be made up.

58
Q

What are the symptoms of cystic fibrosis?

A

Mucus is thicker and builds up. Inflammation reduced by breathing.

59
Q

What is gene therapy?

A

Inserting a functional allele of a particular gene into cells that contain mutated and non-functioning alleles.

60
Q

How could computational biology help us to find new vaccines for the new strain of coronavirus?

A
  1. Base sequence of other coronaviruses held in database. 2. computational analysis allows rapid comparison of RNA sequence of Covid19 with previous strains. 3. Amino acid sequence/ protein structures of antigens also held in database. 4. Computer modelling of protein structures from Covid19 from a base sequence. 5. Computer modelling of vaccines- compare structure of Covid19 antigens. 6. AVP
61
Q

Outline how bacteria can be genetically modified to produce insulin.

A
  1. Adding reverse transcriptase enzyme makes a single strand of C DNA and treatment with DNA polymerase makes a double strand, the gene. 2. Addition of free unpaired nucleotides at the ends of the DNA produces sticky ends. 3. Now, with the help of ligates enzyme, the insulin gene can be inserted into plasmids extracted from E. coli bacteria. These are now called recombinant plasmid, as they contain inserted DNA. 4. E. coli bacteria and mixed with recombinant plasmid and subjected to heat shock in the presence of calcium chloride ions, so that they will take up the plasmids. 5. These are then cultured in large numbers to produce insulin.
62
Q

Explain how the plasmid is prepared.

A

The plasmid is cut using restriction enzymes which bind to specific recognition sites and hydrolyse the phosphodiester bonds in the sugar-phosphate backbone. This produces complementary sticky ends. Anneal gene into the plasmid. Complementary bases from H bonds. Ligase reforms the sugar-phosphate backbone by catalysing the formation of phosphodiester bonds. Now recombinant DNA.

63
Q

Epithelial cells lining in the respiratory tract are replaced every 10-14 days. Why might this mean that treatment has to be repeated at regular intervals?

A

The CFTR gene may become broken down in the cytoplasm by lysosomes. If not integrated into genome, the DNA will not be replicated as the epithelial cell divided. CTFR gene will not be present in the daughter cells.

64
Q

What is the problem with using viruses in somatic gene therapy?

A

Even though the virus has been made safe it still causes an immune response. The patient could become immune to these viruses stopping gene delivery. The virus could insert the gene into part of the gene and which is not important e.g. control cell division, could lead to mutations and cancer. It interferes with normal gene expression.

65
Q

What is the problem with using liposomes in somatic gene therapy?

A

DNA is less likely to be incorporated into nuclear DNA (once inside the cell it might not merge with the nuclear membrane). Less efficient at introducing the gene.

66
Q

What is a problem with both viruses and liposomes in somatic gene therapy?

A

As epithelial cells die and are replaced regularly, the treatment has to be repeated regularly to. This treatment would only help the lung problems caused by cystic fibrosis.

67
Q

Why is altering the genome in the gamete or zygote better?

A

It only has to be treated once/it is permanent. All cells in the body have the replaced working gene. It can be passed on to offspring.

68
Q

Why is germline gene therapy banned in the UK?

A

The descendants can’t give their consent. There are concerns about how the genes may be inserted and if they could disrupt the expression or regulation of other genes which could lead to cancer. It could lead to parents choosing desirable characteristics for their children e.g. designer babies.

Module 6: Genetics, evolution and ecosystems (6 decks)

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