Genetic Technology#

Transgenesis#

1. Identify and obtain the gene of interest.

   There are three ways to identify and obtain the gene of interest

   1. Cut from chromosomal DNA using restriction endonuclease
   2. Made by making a single-stranded complementary DNA (cDNA) from mRNA by reverse transcriptase and then converting the cDNA into a double-stranded DNA molecule by DNA polymerase
   3. Synthesised chemically from nucleotides

2. Make multiple copies of the gene/clone and amplify the gene by using the polymerase chain reaction (PCR). (PCR：详见下)

3. Cut the two ends of the DNA with the gene of interest using restriction enzyme/restriction endonuclease限制性内切酶, forming sticky ends粘性末端.

   restriction enzymes recognise specific short DNA sequences and cut at these points, form the sticky ends

   For example, the restriction enzyme BamHI consistently cuts DNA at a specific recognition site

   • 5’ to 3’ strand: GGATCC (this restriction enzyme cut at this location)
   • 3’ to 5’ strand: CCTAGG (complementary sequence)

   It is a palindrome (回文), meaning the sequence reads the same in both directions (when reading 5’ to 3’ on each complementary strand).

4. Cut plasmid, a vector 载体, with the same restriction enzyme, forming complementary sticky ends.

   The vectors can be: plasmids, viruses, liposomes, naked DNA

5. Insert the gene into cut plasmid using DNA ligase (DNA连接酶), form a recombinant plasmid.

   DNA ligase - Catalyse the formation of phosphodiester bonds

   The sticky ends from the gene of interest and the cut plasmid are complementary, and form hydrogen bonds between them.

6. Insert recombinant plasmids into bacteria.

   1. Treat bacteria with a solution of Ca²⁺ ions
   2. Apply heat shock (~42°C) or use electroporation 电穿孔 to increase chances of plasmids passing through cell surface membrane
   3. Bacteria that take up recombinant plasmids are said to be transformed.

7. The transformed bacteria are identified (详见下), often by using marker genes which are added to the vector and inserted close to gene of interest.

8. Cultivate培养 and clone transformed bacteria containing recombinant plasmids in a fermenter; the gene of interest is expressed in bacteria to make proteins.

Summary of the complete process#

1. Isolate mRNA for the gene required. 分离目标基因
2. Use reverse transcriptase to produce single-stranded cDNA. 合成单链cDNA
3. Use DNA polymerase to produce double-stranded cDNA. 合成双链cDNA
4. Use an enzyme to add short length of single-stranded DNA to form sticky ends. 添加sticky ends
5. Use a restriction enzyme to cut plasmids. 切开plasmid
6. Mix the double-stranded DNA with plasmids. 往plasmid中添加新的基因
7. Form recombinant plasmids by complementary base pairing.
8. Use ligase to seal the sugar–phosphate backbone of the recombinant plasmid. 重新把plasmid连回去
9. Insert the plasmid into a host bacterium. 重新把plasmid放回bacterium中
10. Clone the modified bacteria and harvest the recombinant protein. 批量生产

Gene therapy#

Treatment of a genetic disorder by introducing functioning normal genes into cells of an organism.

Examples of genetic diseases treated with gene therapy

• severe combined immunodeficiency (SCID)

  autosomal recessive condition - T-lymphocytes are affected

  To protect a patient from infections, during gene therapy, a virus transfers a normal dominant allele for ADA into T-lymphocytes.

  This is not a permanent cure as the T-lymphocytes are replaced naturally overtime.

• inherited eye disease LCA

  blindness; Leber congenital amaurosis

  usually an autosomal recessive condition

Use viruses or liposomes, or naked DNA as the vectors to transmit the normal DNA

How the genetic disease may be treated using gene therapy:

1. normal allele is inserted into vector ;;
2. the vector liposomes in aerosol fuses with host cell ;;
3. OR use virus (harmless virus) as the vector ;;l
4. This is only a short term effect, the treatment has to be repeated ;;

Considering different vectors:

• Naked DNA

  • has to be injected into target cell / lack of organ-specific delivery ;

  • low efficiency of cellular uptake ;

• Viruses

  • rapidly broken down ;

  • small packaging capacity / only small amount of DNA can be carried ;

  • low probability of integration (into host genome) ;

  • cause mutations in host DNA / (gene) insertion disrupts gene function / insertional mutagenesis ; and may cause cancer and other side effects ; e.g. infection; can insert normal healthy allele randomly into host DNA and in wrong place

• Liposomes

  • low ability to, add DNA / genes, into target cells (genome) / low transduction efficiency ;

The social considerations of using a retrovirus for gene therapy:

• the retrovirus may disrupt other gene
• the retrovirus must not cause…
  • cancer
  • infection
  • allergic response

Gene editing#

A form of genetic engineering involving the insertion, deletion or replacement of DNA at specific sites in the genome of an organism using a method such as the CRISPR/Cas9 system

CRISPR/Cas9 system developed from a mechanism used by some bacteria to defend themselves against viral infections

• CRISPR is a group of base sequences that code for short lengths of guide RNA that direct an endonuclease enzyme known as Cas9 towards specific base sequences.
• CRISPR-Cas9 system can be used to repair a gene that has a harmful mutation.

Involve modification of the existing DNA of an organism rather than the insertion of DNA from another organism.

Why sometimes gene editing is more suitable as a potential cure for some disease:

• the disease may be caused by a dominant ellele
• adding a normal recessive allele would not work
• gene editing can alter the mutant allele / DNA

Gel Electrophoresis 凝胶电泳#

This is a technique that is used to separate DNA fragments of different lengths.

DNA molecules are negatively charged

The gel contains many pores, smaller molecules move through the gel faster than large molecules:

参考来源：【实验动画化】四分钟秒懂 DNA 凝胶电泳_哔哩哔哩_bilibili

1. Agar/agarose gel is made. DNA is fragmented by restriction enzyme and loaded into wells at the negative end of agar/agarose gel. 准备凝胶和DNA片段
2. Gel is submerged in buffer solution. 将凝胶放入缓冲液中
3. Phosphate groups of DNA are negatively charged. When direct current is applied, DNA is attracted/moves to anode/positive electrode. 通直流电时向正极移动
4. Larger fragments of DNA move more slowly due to gel resistance/impedance. 片段越小移动越快越远
5. DNA fragments of different sizes separate due to electric field. 发生分离
6. DNA was stained so that UV light can be used to observe DNA banding pattern. 通过UV light观察染色了的片段

可以通过和已知长度的片段（marker）来估算右边移动的marker长度。

Electrophoresis can also be used to separate protein / RNA pieces with different lengths.

Genetic Fingerprinting#

Belong to genetic/DNA profiling 基因谱分析

Based on the fact that some regions of DNA have highly variable repetitive sequences (e.g. Variable Number Tandem Repeats, VNTR 可变数目串联重复序列) 在DNA中有些部分是独一无二的（除了同卵双胞胎）

• unique to each individual - These unique DNA sequences can be used to identify individuals, similar to how fingerprints are used for identification.
• except for identical twins.

The greater the number of repeats, the longer the fragment of DNA.

Electrophoresis经常在做genetic fingerprinting的时候使用：

1. Extract DNA with VNTR sequences. Amplify DNA by PCR.
2. Agar/agarose gel is made. DNA is fragmented by restriction enzyme and loaded into wells at the negative end of agar/agarose gel.
3. Gel is submerged in buffer solution.
4. Phosphate groups of DNA are negatively charged. When direct current is applied, DNA is attracted/moves to anode/positive electrode.
5. Larger fragments of DNA move more slowly due to gel resistance/impedance.
6. DNA fragments of different sizes separate due to electric field.
7. DNA was stained so that UV light can be used to observe DNA banding pattern.

Microarray 微阵列#

Also known as a gene or DNA chip based on a small piece of glass or plastic usually 2 cm².

参考来源：DNA微阵列是什么？它是如何比较2种细胞，进而分析，哪些基因是激活的？哪些是抑制的？_哔哩哔哩_bilibili

Microarrays are used to find out which genes are expressed within cells and to identify the genes present in an organism’s genome. 用来比较哪些基因被activated激活和哪些基因被repressed抑制

A microarray contains thousands of tiny spots, each of which contains a single-stranded DNA probe 探针 from a particular known gene and unique to the gene.

Each ssDNA probe has a complementary base sequence to a gene being detected. 这个每个探针只对一种基因有效，当这个基因被激活时，探针也会相应地做出反应。

How to do microarray to find out expression of genes?

1. Extract and obtain mRNA from cell. Use reverse transcriptase to carry out reverse transcription of mRNA to produce single stranded cDNA. Add fluorescent label to cDNA. 将mRNA制作成带染色的cDNA（两个不同的样本需要标记不同的颜色）
2. Microarray has ssDNA probes, each of which is from a particular known gene.
3. Add cDNA to microarray and cDNA hybridises to ssDNA probes by complementary base pairing (reverse transcription). cDNA与探针杂交（互补配对）
4. Wash off excess cDNA and expose microarray to UV light. 去除（洗掉）多余没有bind上的cDNA
5. Fluorescence shows the expressed genes. 把板子放在紫外灯下看。有荧光的地方，就说明这个基因在细胞里表达了。
6. Intensity of fluorescence gives quantitative measure/shows level of gene expression. 越亮说明基因表达得越多

TIP

cDNA不是基因本身，它是通过mRNA反转录来的。这意味着——我们看到的荧光，其实是细胞当时正在“喊话”的基因（mRNA），而不是深藏在细胞核里“沉睡”的DNA。

我们看到的荧光（cDNA），不是来自细胞核里永远存在的那份“死”DNA原稿，而是来自细胞质里那份“活”的、临时的工作单（mRNA）。

之所以要强调这个“陷阱”，是因为很多初学者会误以为：“基因在DNA里，我测到DNA就等于基因在表达”。但这是错的！ 因为：

• 即使某个基因的DNA永远存在于细胞核里（比如所有人的癌细胞里都有“癌基因”的DNA），
• 但只要它没有转录成mRNA（没有复印成工作单），
• 那么在Microarray芯片上就绝对不会有荧光，这个基因就被认为是“关闭/抑制”状态。

- DeepSeek

NOTE

Describe how microarray analysis can detect differences in the expression of many genes when comparing two samples, such as the offspring of wild and captive-bred fish. any five from:

1. obtain mRNA from wild and captive bred fish / AW 分别从两个samples中提取mRNA信息 ;
2. reverse transcription of mRNA to produce cDNA 制作cDNA ;
3. add fluorescent label to (c)DNA 添加染色物质 ; I colour A dyes / markers / tags
4. (microarray has) ssDNA probes 需要提到microarray上最重要的ssDNA probe ;
5. each from a, different / known, gene 每个probe能检测不同的已知gene ;
6. (c)DNA hybridises / AW, to, probes / ssDNA (on microarray) ;
7. fluorescence shows the expressed genes 如果gene表达了出来，那么就会有荧光的颜色 ;
8. intensity of fluorescence (shows level of gene expression) 颜色越强烈，gene表达得越多 ;
9. compare, fluorescence / gene expression (between fish groups) 对比两个samples中的gene表达情况 ;
10. AVP ; e.g. ref. to washing off excess (c)DNA (only), after hybridisation UV light / laser scanning (to record fluorescence pattern)

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(a) Outline the steps required to prepare the genome of an individual so that the genome is ready for analysis using a microarray chip.

这个问题问的是怎么准备这个microarray上的所有基因样本，而不是参与“表达性”测试的cDNA。

基因组（Genome） = 细胞核里所有的DNA（包括编码的、不编码的、开启的、关闭的）

any three from:

1. ref. to isolate / extract, DNA, from, cells / tissue / named 提取DNA ;
2. ref. to cut DNA, into small fragments / using restriction enzymes 分割DNA ;
3. ref. to denature / AW, into single-stranded DNA 通过denaturation，把double-stranded DNA变成single-stranded DNA（以便在后面和参与测试的cDNA结合到一起） ;
4. add fluorescent, tag / marker / dye 染色 ;

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(i) Outline how microarrays are used in the analysis of genomes. any four from:

1. obtain single-stranded DNA ;
2. label DNA with fluorescent, dye / tag / label ;
3. probes on, chip / microarray ;
4. each probe is unique to a particular, SNP / gene / allele ;
5. DNA (added), binds / hybridises, to probes ;
6. wash off excess DNA (after hybridisation) ;
7. (view under) UV light / laser scanner / high resolution digital camera ;
8. fluorescence indicates presence of, SNP / gene ; R gene expressed

Bioinformatics 生物信息学#

Bioinformatics - use computer softwares to analyse the biological informations, there is a database contains the information about DNA sequences.

Large databases contain DNA base sequences and amino acid sequences of different species and organisms, allowing data to be shared.

The role of bioinformatics following the sequencing of genomes of humans and parasites:

• to allow the data to be shared
• can predict the amino acid sequence
• provide analytical tool , o.g. BLAST
• compare the base sequences

NOTE

Outline how sequencing the genome of *Plasmodium* and the use of bioinformatics can suggest new targets for anti-malarial drugs. [max 3]

1. identify / recognise, gene(s) ; A find where genes are
2. predict, primary structure / amino acid sequences, of proteins ;
3. predict 3D structure of proteins ; A tertiary
4. identify / predict, functions of proteins (from 3D structure) ;
5. ref. to drug to, bind with / block activity of / disrupt structure of, protein / enzyme ; A drug specific to protein I denature, protein / enzyme
6. drug prevents, transcription / expression, (of gene) ; I gene editing

The advantages of using theoretical models in research rather than testing possible drugs in the laboratory:

• cheaper 更加便宜
• faster 更快
• no need to test on animals / humans 可以避免道德问题
• no need for laboratories and equipment 不需要使用器材

但是使用理论模型不能完全代替试验操作，因为：

• cannot assume the predictions are correct

• there may some clinical traits / side effects

• have to make sure the dosage is safty

  theoretical modelling will not give information on doses

Genetic screening 遗传筛查#

Analysis of a person’s (embryo’s, fetus’ or adult’s) DNA to check for the presence of one or more alleles that are associated with disease.

Genetic screening helps provide early diagnosis

DNA sample can be obtained by:

• Taking tissue samples from adults
• Embryo biopsy 胚胎活检 - embryo produced by in-vitro fertilisation; one or several cells are removed;
• Chorionic villus sampling 绒毛取样 or amniocentesis 羊膜穿刺术

遗传筛查的使用例子：

• Breast cancer - Genes BRCA1 and BRCA2

• Huntington’s disease - HTT gene

• Cystic fibrosis 囊性纤维化 - CFTR gene

  Cystic fibrosis - a genetic disease caused by recessive alleles of the CFTR gene

使用遗传筛查的好处：

• if negative - reduce worry
• if positive
  • change the lifestyle
  • have early treatment
  • informed decision about having children

Genetic technology’s summary#

Outline the different ways in which genetic technology can be applied to medicine, with reference to named diseases. [7]

Genetic engineering (recombinant DNA technology):

• make drugs from genetic modified bacteria / yeast
• e.g. making human insulin (for diabetes)
• e.g. making factor VIII (for haemophilia)
• e.g. making adenosine deaminase (for **SCID)

Genetic screening:

• detect BRCA1 amd BRCA2 (for breast cancer)
• detect Huntington’s disease
• detect CFTR for cystic fibrosis

Gene therapy:

• add normal alleles into the tissue of person
• e.g. to treat eye diseases
• e.g. to treat SCID