What Is a Diagnostic Molecular Scientist?

Some fertilized eggs (germplasm) or mothers are affected by environmental or genetic influences, causing harmful changes in the next-generation genome, and (physical) diseases. Genetic diagnosis and genetic analysis can only be confirmed. Also called DNA diagnosis or molecular diagnosis. Using the current human understanding of the genome and molecular genetics data, check the molecular structure level and expression level to diagnose common genetic diseases or family genetic diseases.

Chinese name: Genetic diagnosis
Foreign name: Genetic diagnosis

Alias: DNA or molecular diagnosis
Classification: Direct genetic diagnosis, indirect genetic diagnosis
Fundamental: Nucleic acid molecule hybridization

Introduction to genetic diagnosis

Genetic diagnosis The diagnosis of single-gene genetic diseases mainly depends on clinical observation and a series of chemical tests, but biochemical tests require body fluids or cells with corresponding gene expression products, and have an understanding of gene products or metabolic abnormalities. However, for most genetic diseases, In other words, this understanding is far from being reached. The ideal diagnostic method is to directly analyze the patient's genes or DNA itself, because this analysis is free of the above-mentioned limitations. Nuclei cells in various tissues of the body have a full set of genomic DNA, which can be used as materials for analysis without having to consider expression.

Genetic diagnostic classification

Genetic diagnosis can be divided into two categories:

Genetic diagnosis

Check the abnormality of the pathogenic gene directly. It usually uses the gene itself or the DNA sequence next to it as a probe, or amplifies the product by PCR to detect no abnormalities, such as mutations, deletions, and degradation of the gene and its properties. This is called direct genetic diagnosis, and it applies to known genetic abnormalities. disease;

Genetic diagnosis

SSCP, AMP-FLP and other technologies can be used for linkage analysis.

Basic principles of genetic diagnosis

Nucleic acid molecular hybridization is one of the most basic methods of genetic diagnosis. The basic principle of gene diagnostic technology is that single strands of complementary DNA can be combined into double strands under certain conditions, that is, they can be hybridized. This binding is specific, that is, strictly based on the principle of base complementarity, it can be performed not only between DNA and DNA, but also between DNA and RNA. Therefore, when a nucleic acid sequence of a known gene is used as a probe to make contact with the denatured single-stranded genomic DNA, if the bases of the two are perfectly matched, they are complementary to form a double-strand, thereby indicating the genomic DNA being tested It contains known gene sequences. It can be seen that there are two necessary conditions for genetic testing. One is the necessary specific DNA probe; the other is the necessary genomic DNA. When both are denatured to a single-stranded state, molecular hybridization can proceed.

I. Gene probe A probe is a specific nucleotide sequence that is complementary to the gene of interest or DNA. It can include the entire gene or just a part of the gene; it can be the DNA itself or the Transcribed RNA.

1. Sources of probes There are three types of DNA probes according to their source: one is from the relevant gene itself in the genome, called a genomic probe; the other is to obtain mRNA from the corresponding gene transcription, and then reverse it The recorded probe is called cDNa probe. Unlike genomic probes, cDNA probes do not contain intron sequences. In addition, a DNA fragment complementary to a gene sequence with a small number of bases can be artificially synthesized in vitro, which is called an oligonucleotide probe.

Genetic diagnosis 2. Preparation of probes Molecular mutations require a large number of probe copies, which are typically obtained by molecular cloning. Cloning is the use of asexual reproduction methods to obtain a large number of copies of the same individual, cell or molecule. When preparing genomic DNA probes, a genomic library should be prepared first, that is, the genomic DNA is interrupted or incomplete hydrolysis is performed with restriction enzymes to obtain many random fragments of various sizes. These fragments are recombined into a carrier (phage) , Plasmids, etc.), and then transfect the latter with appropriate host cells such as coliform bacteria. At this time, many clonal plaques carrying different DNA fragments can be obtained on solid media, and can be obtained by in situ hybridization. The clones containing the gene fragment of interest were screened out, and then a large number of probes were prepared by cell expansion.

In order to prepare a cDNA probe, the corresponding mRNA needs to be separated and purified first, which is relatively easy to do from tissues and cells containing a large amount of mRNA, such as preparing alpha or beta globin mRNA from hematopoietic cells. With mRNA as a template, complementary DNA (ie, cDNA) can be synthesized under the action of reverse transcriptase. The cDNA and the coding region of the gene to be tested have exactly the same base sequence, but the introns are already in the Cut off during processing.

Oligonucleotide probes are synthetic, complementary to the DNA of known genes, and can range from a dozen to tens of nucleotides in length. If only the amino acid sequence of a protein is known, the nucleotide sequence can also be deduced according to the amino acid code and synthesized by chemical methods.

3. Labeling of probes In order to determine whether the probes hybridize to the corresponding genomic DNA, it is necessary to label the probes in order to obtain an identifiable signal at the binding site. Usually, a certain nucleotide of the probe is labeled with a radioisotope 32P Alpha phosphate. However, in recent years, some methods have been developed using non-isotopes such as biotin, digoxigenin, and the like as labels. But they are not as sensitive as isotopes. Non-isotopic labeling has the advantage of longer storage time and avoids isotope contamination. The most commonly used probe labeling method is nicktranslation.

Probes can also be labeled with random primers, that is, a small 6-nucleotide fragment of random DNA is added to the denatured probe solution as a primer. When the latter is complementary to single-stranded DNA, it is continuously added based on the principle of base complementarity. Adding an isotope-labeled single nucleotide to its 3'OH end can also obtain DNA probes with higher specific radioactivity.

2. Restriction endonuclease, also referred to as restriction enzyme or endonuclease. They are an important class of tool enzymes for genetic engineering and genetic diagnosis. Their discovery and application provide the necessary means to isolate the target gene from the genome. Restriction enzymes can specifically recognize and cut specific nucleotide sequences, and cut double-stranded DNA into smaller fragments. After digestion, the target gene may be completely or partially stored on a certain DNA fragment and isolated.

Restriction enzymes are mainly derived from prokaryotes, and are a group of enzymes that can hydrolyze the phosphodiester bonds of DNA. Hundreds of restriction enzymes have been discovered so far, divided into three categories. The second category is mainly used in genetic engineering. Restriction enzymes are named after their source.

Each restriction enzyme typically recognizes and cleaves a 4-6 nucleotide sequence, called a restriction site or cut point. There are two ways for restriction enzymes to cut double-stranded DNA, and there are two types of ends. The first is staggered cleavage, that is, the cut points of the two strands are not at the same level but are separated by several bases, so the fracture produces two small segments of itself. Complementary single strands. These ends are easily complementary and are called cohesive terminus; the second is a flat cut.

The above characteristics of restriction enzymes have important uses in genetic engineering and genetic diagnosis: First of all, regardless of the source of the DNA, the sticky ends generated after cutting with the same endonuclease are easy to reconnect, so it is easy to connect humans and bacteria or humans. It is connected with any two DNA fragments of the plasmid, that is, recombination, which is the basis of recombinant DNA technology. The human genome is large, and the genes cannot be analyzed without cutting. Restriction enzymes can cut the genome at specific locations, that is, the cut is not random, so the same set of fragments of different lengths is obtained from the genome of each cell. These fragments, which may contain a gene, can be separated by electrophoresis and studied. Due to the specificity of the restriction enzyme, if the base of the recognition site is changed, the restriction enzyme will no longer be able to cut; similarly, the change of the base may also cause the appearance of a new acid cleavage site. In the human genome, these two conditions are very common, and the disappearance or appearance of the cut point will affect the length of the obtained DNA fragment, which is expressed as restriction fragment length polymorphism (RFLP), which is used in the linkage diagnosis of genes. It is extremely important.

3. Restriction fragment length polymorphisms Two sets of haploid DNA of a person are not exactly the same, and generally one of every 100-500 base pairs is different. In other words, if two sets of genomic DNA (3.2 x 109bp each) are arranged, there are 10 million differences on average, and they are mostly located in the intron sequence. In fact, no two individuals in the population have the same genomic DNA, except for the single egg twins.

Although DNA polymorphisms can be detected by DNA sequencing, digestion with restriction enzymes is the most commonly used detection method.

1. RFLP due to the mutation of the base may cause the disappearance of the digestion point or the emergence of a new digestion point, which may cause different individuals to use the same restriction enzyme to cut the length of the DNA fragment. Differences in DNA fragment length are called restriction fragment length polymporphism (RFLP). RFLP reflects the common heritable variation of DNA nucleotides between individuals, which is inherited in Mendelian way. RFLP can be detected by Southern blot hybridization. When RFLP is detected by Southern hybridization, if the probe crosses the cut point, both of the cut fragments can hybridize with the probe, showing two hybridization bands.

2. Two-point RFLP

(1) Point polymorphism: It is an RFLP caused by the appearance or disappearance of the enzyme cut point caused by the change of a single or a few bases. The aforementioned RFLP falls into this category. They belong to the classic RFLP. Hundreds of such polymorphic sites have been found in the human genome.

(2) Variable number tandem repeats (VNTR): RFLP caused by the above-mentioned classic single base substitution generally can only detect two forms of heterozygosity, namely "have" or " "No" restriction site, and the heterozygous frequency of each site in the population usually does not exceed 50%. When the test individual is homozygous, the required polymorphic information cannot be obtained using RFLP . In addition, RFLPs of this type are currently found to be limited and unevenly distributed throughout the genome.

However, there is another type of DNA repeats in the human genome, called small satellite DNA. They are widely distributed, and each unit is usually only 16-28bp long, but the number of repetitions is highly variable in the population. When a restriction enzyme is used to cut the VNTR region, as long as the restriction site is not in the repeat region, it is possible to obtain fragments of different lengths different from small satellite DNA. Another type of repeat sequence is satellite DNA. Their basic sequences are 1 to 6 bp, such as (TA) n, (CGG) n, etc., which are usually repeated 10 to 60 times and are highly polymorphic.

VNTR has a high degree of variability and is also inherited according to the Mendelian method, so it is a good genetic marker. Because of its many types and wide distribution in the genome, it is increasingly used in genetic linkage diagnosis.

Genetic diagnosis

The genetic abnormalities of various genetic diseases are different, and the same genetic disease can also have different genetic abnormalities, but these abnormalities can be roughly divided into two types of gene deletion and mutation. The latter include single base substitutions, minor deletions or insertions. Some genetic diseases discovered in the 21st century are caused by the increased sequence of trinucleotide repeats in genes. Depending on the type of genetic abnormality, different diagnostic methods can be used. For example, gene deletion can be hybridized with gene probes and detected directly by PCR amplification; point mutations can be directly checked with allele-specific ASO probes and SSCP. Analysis of family members is generally not required. However, it is necessary to know the nature of the genetic abnormality and to confirm the relationship between the abnormality and the disease. However, due to the genetic heterogeneity of many diseases and most inherited genetic abnormalities are unknown, although the number of diseases that can be directly diagnosed in the 21st century is increasing, they are still relatively limited.

Genetic diagnosis The genes of many genetic diseases have not been isolated or cloned, or genetic abnormalities are unclear, so diagnosis cannot be made based on the nature of the mutation. However, if a family analysis can prove that a DNA marker (whether an allele or a polymorphic site or fragment) is linked to a disease-causing gene, all members carrying the marker may carry the disease-causing gene, thus Make an indirect linkage analysis diagnosis.

Selection of diagnostic methods for genetic diagnosis

When applying linkage analysis for diagnosis, the following issues should be noted: Recombination between genes and DNA markers may occur. Therefore, the accuracy of linkage analysis depends on how closely the DNA is linked to the disease-causing gene. The closer the linkage, the higher the reliability. Therefore, a marker that is as close to the disease-causing gene as possible, that is, a tightly linked marker, or multiple genetic markers should be used to minimize May reorganize. Because there may be recombination, linkage analysis cannot fully determine the presence or absence of the pathogenic gene, but rather indicates the probability or probability of its existence. If the marker distance gene is 5 cM, that is, the recombination rate is 5%, then there is a 5% error in making a diagnosis, that is, only 95% can make a positive or negative conclusion. Heterozygosity of selected genetic markers in the population. If the heterozygosity of the marker is low in the population, that is, most individuals are homozygous, then the marker is not useful. Because key members in the family cannot provide information on which chromosome the pathogenic gene is on, often making linkage analysis impossible, so a marker with high heterozygosity in the population needs to be selected. In linkage analysis, sometimes only one polymorphic site can be analyzed to distinguish the chromosomes with disease-causing genes from normal chromosomes. This is usually because the parents of the key member, such as the patient, are homozygous and cannot provide the necessary information. At this time, more polymorphic sites can be analyzed at the same time, that is, haplotype analysis. A haplotype is a combination of the states of two or more polymorphic sites on a chromosome. After all, the probability of homozygosity at multiple loci on two chromosomes is very small, so haplotypes can help distinguish two autosomes and track the segregation of disease-causing genes.

Gene Diagnostic Techniques

Genetic diagnosis review

When the cell's genomic DNA is cut with a specific endonuclease such as Eco R, gene diagnosis is cut where GAATTC is found, and many fragments of a certain length but not equal to each other are obtained. The genes or DNA fragments that need to be analyzed and separated are On one of those particular clips. However, it is difficult to analyze many DNA fragments of different lengths mixed together. Therefore, they must first be separated by size (length), which can be accomplished by means of gel electrophoresis. During electrophoresis, the smaller the molecular weight, the faster the migration, and the larger the fragment, the slower. Therefore, at the end of electrophoresis, a continuous band spectrum (smear) can be obtained, and a specific fragment obtained from the genomes of many cells will be in the same position because of the same length, which is conducive to detection. But the gel is brittle and inconvenient to handle. British scientist Southern pioneered the above-mentioned difficulties.

Southern Genetic Diagnostic Southern Blot

The basic principle of Southernblot is: the nitrocellulose membrane or nylon filter has a strong ability to adsorb single-stranded DNA. After electrophoresis, the gel is subjected to DNA denaturation treatment, covered with the above-mentioned filter, and then a layer of dry absorbent water is pressed on it. Paper, by virtue of its suction effect on the deep salt solution, the single-stranded DNA on the gel will be transferred to the filter. The transfer is in situ, ie the position of the DNA fragment remains the same. After the transfer was completed, the DNA that had been baked at 80 ° C was fixed on the membrane in situ.

After a specific gene fragment has been transferred to the membrane in situ, it can be hybridized with an isotope-labeled probe and the hybridization signal can be displayed. Hybridization is usually performed in a plastic bag. The hybridization filter is placed in the bag. After adding the hybridization solution containing the denatured probe, the single-stranded probe DNA and the single-stranded gene DNA molecule fixed on the membrane are alkalized at a certain temperature. The basis-to-complementary principle is fully combined. Binding is specific, for example, only beta globin gene DNA can bind to beta globin probes. After hybridization, the uncombined probe on the membrane was washed away, the X-ray film was coated on the membrane, and autoradiography was performed in the dark in the dark. The location of the DNA fragment incorporating the isotope-labeled probe will show a black hybridized band, and a deletion or mutation of the gene may cause the deletion or position change of the band.

Molecular hybridization is the basis of gene detection. In addition to blot hybridization, there is also dot hybridization. That is, the DNA sample is directly spotted on the nitrocellulose filter after hybridization, and then hybridized with the probe, or the cells or viruses are spotted on the membrane, and the colonies or plaques are adsorbed on the membrane in situ, and then denatured, and then hybridized. Dot hybrids are mostly used for pathogen genes, such as those of microorganisms, but they can also be used to examine DNA sequences in the human genome.

Genetic diagnosis polymerase chain reaction

In the 21st century, there has been a revolutionary breakthrough in gene analysis and genetic engineering technology, which is mainly due to the development and application of polymerase chain reaction (PCR). The application of PCR technology can enable specific genes or DNA fragments to be amplified hundreds of thousands to millions times in vitro in just 2-3 hours. The amplified fragments can be viewed directly by electrophoresis or used for further analysis. In this way, a small number of single-copy genes need not be observed by isotopes to increase their sensitivity, but can be directly observed after being amplified to a million times, and the diagnosis that originally took one or two weeks can be shortened to several hours.

First, an oligonucleotide with a length of about 17-20 bases should be synthesized as a primer in accordance with the base sequence of the 5 'and 3' ends of the DNA to be detected, and then the DNA to be detected is denatured. Add four single nucleotides (dNTPs), primers, and thermostable polymerase. At lower temperatures, the primers will be renatured with the DNA strand to be amplified, and the polymerase will then use the nucleotide material in the solution to continuously extend to synthesize new complementary strands. In this way, one DNA double strand will be Into two double strands. If you continue to cycle for 20 to 40 cycles in the order of denaturation (92-95 ° C) renaturation (40-60 ° C) primer extension (65-72 ° C), a large number of DNA fragments can be obtained. Theoretically, 20 cycles can make the DNA amplified by 2n, which is more than 1 million times. The PCR reaction has strong specificity and high sensitivity, and a very small amount of DNA can be used as an amplification template to obtain a large number of amplified fragments. Hair, bloodstains, and even individual cell DNA can be used for PCR amplification. Therefore, it is used for the detection of pathogen DNA, the detection of tumor residual cells, the identification of criminal or individual genetic material, and the genetic diagnosis of genetic diseases.

PCR diagnosis is now available for a range of genetic diseases. If the disease is caused by a gene deletion (such as alpha thalassemia), designing a pair of primers for amplification at both ends of the deletion will not result in amplification products or only shortened amplification products. If the disease is caused by a point mutation, and the location and nature of the mutation are known, then design the primers to include the mutation site, because the mutated bases do not match, and there is no amplified fragment; or A wrong nucleotide is designed at the 3 'end to pair with the mutated nucleotide. As a result, normal primers cannot be amplified, but incorrect primers can be used to amplify the existence of mutations.

Since the 21st century, there have been many new developments in PCR technology, with increasingly expanding uses. For example, RT-PCR using RNA as a template and then amplifying by reverse transcription; changing the concentration of the two primers to make them 100 times different, resulting in a large number of single-stranded products, called asymmetric PCR, which can be used for sequence analysis ; Multiple PCR with multiple primers in one reaction to detect multiple sites at the same time.

Genetic Diagnostic Amplified Fragment Length

The polymorphism of small satellite DNA and microsatellite DNA can be detected by PCR amplification and electrophoresis, and used for linkage analysis of pathogenic genes. This diagnostic method is called amplified fragment length polymorphism (amplified fragment length polymorphism (Amp-FLP) linkage analysis. After PCR amplification, the difference between the products or alleles is sometimes only a few nucleotides, so polyacrylamide gel electrophoresis is required for separation and identification. This method is mostly used for linkage analysis with unknown mutation properties.

Allele-specific

Oligonucleotide probe diagnostics When the mutation site and nature of a gene are fully understood, an isogenic-specific oligonucleotide probe (ASO) can be synthesized and diagnosed using isotopic or non-isotopic labeling. Probes are usually about 20 bp in length. When detecting point mutations, two types of probes need to be synthesized. One is completely consistent with the normal gene sequence and can stably hybridize with it, but cannot hybridize with the mutant gene sequence. The other is consistent with the mutant gene sequence and can interact with the mutation. Gene sequences can be stably hybridized, but they cannot be stably hybridized with normal gene sequences. In this way, genes with mutations in only one base can be distinguished.

PCR can be combined with ASO, that is, PCR-ASO technology, that is, in vitro amplification of relevant fragments of genes containing mutations, and then dot hybridization with ASO probes, which greatly simplifies the method, saves time, and requires only a small amount Genomic DNA is ready.

Single-strand conformation polymorphism

Single-strand conformation polymorphism (SSCP) refers to the difference in conformation of single-stranded DNA due to different base sequences. This difference will cause the same or similar length of single-stranded DNA to have different electrophoretic mobility, which can be used for Detection of single base substitutions, minor deletions or manuscripts in DNA. When using SSCP method to detect gene mutations, a pair of primers are usually designed for PCR amplification near DNA fragments suspected of being mutated, and then the amplified products are denatured with formamide or the like, and electrophoresed on a polyacrylamide gel. Differences in DNA conformation will manifest as differences in the position of the electrophoretic bands, which can be used for diagnosis.

The PCR-SSCP method has the advantage of being able to quickly and sensitively detect the presence or absence of point mutations or polymorphisms, but to clarify the nature of the bases of mutations, sequence analysis is required.

Examples of genetic diagnosis of genetic diseases

1. Diagnosis of genetic deletion (1) Genetic diagnosis of alpha thalassemia: Alpha thalassemia is mainly caused by gene deletion, and the number of missing genes can be 1-4. The normal genome was cut with BamHI to get a 14 kb fragment. When the alpha gene was deleted, the cut point was shifted to the 5 'end to obtain a 10 kb fragment. Therefore, when the gene probe is used for Southern hybridization with genomic DNA (Figure 13-8), a 14 kb and a 10 kb band can be seen in thalassaemia 2, and a double 14 kb band can be seen in normal people. Alpha thalassemia 1 saw a single copy of a 14 kb band. In hemoglobin H disease, there was only a 10 kb band. In Barts' edema, there was no hybrid band.

A simpler method is to perform spot hybridization directly with an alpha probe. After autoradiography, the diagnosis of alpha thalassemia can also be made according to the difference in the depth of the spots. A simpler method is PCR diagnosis, that is, design a pair of primers in the alpha gene deletion range, and then PCR amplify the fetal DNA. If it is a Barts edema fetus, there are no amplification products and no banding after electrophoresis, so that Abortion is recommended, but this method cannot diagnose other types of thalassaemia (unless primers are designed for PCR).

(2) DMD / BMD-deficient diagnosis: DMD / BMD is a fatal genetic disease involving the X-linked recessive inherited neuromuscular system (see Chapter 4). About 70% of DMD / BMD are missing. This gene is very large, and deletions can occur in different locations, so as many PCR primers as possible (multiplex PCR) should be used for detection. If banded deletions are found after the electrophoresis of the amplified product, diagnosis can be made and the deletions can be located (Figure 13-9). When performing prenatal diagnosis, you can usually determine the proband by testing the relevant members of the family. The deleted region is then targeted for PCR amplification, including both ends of the deleted portion, to determine whether the fetus or the affected child has also obtained the same gene deletion, but the non-deleted type cannot be detected by this method.

2. Genetic diagnosis of point mutation genetic disease 2 (1) Genetic diagnosis of sickle cell anemia: It is known that the mutant gene is the 6th codon encoding -globin chain changed from GAG to GTG, thus replacing valine Glycine can therefore be diagnosed as follows.

1) RFLP diagnosis: The recognition sequence of the known restriction enzyme Mst is CCTNAGG, which can cut the CCTGAGG sequence in the normal chain, but cannot cut the mutated CCTGTGG (A T). In this way, because the mutation eliminates an cleavage point and changes the length of the endonuclease, the normal A and S can be distinguished by electrophoresis.

2) ASO probe diagnosis: Since the mutation site and nature are fully understood, oligonucleotide probes can also be synthesized and diagnosed by 32P standardization. At this time, two probes need to be synthesized, one is completely consistent with the normal A gene sequence and can stably hybridize with it; the other is consistent with the mutant gene sequence and can be stable with the S gene, but cannot be hybridized with the normal A gene . Based on the hybridization results, mutations in the S gene can be detected.

Since the advent of PCR technology, ASO diagnosis has been improved by firstly amplifying a gene fragment of about 110bp in length by PCR and then hybridizing with ASO probes. This can reduce the amount of DNA of the target gene and reduce non-specific signals when hybridizing with genomic DNA.

3 Adult polycystic kidney disease (APKD) is an autosomal dominant genetic disease with a high incidence. There is a carrier of a disease-causing gene in about 1,000 people. The disease is relatively late, mostly after 30 years of age, mainly due to multiple cysts in the kidney and liver. Clinical manifestations include backache, proteinuria, hematuria, hypertension, pyelonephritis, kidney stones, etc., which can eventually lead to renal failure and uremia. . The disease gene is located at 16p13, which is adjacent to the 3 'end of the alpha globin gene, but the pathogenic gene has not been cloned, and the biochemical properties of the gene product and the pathogenesis of the disease have not yet been elucidated. Therefore, linkage analysis can only be used for pre-onset and prenatal diagnosis of genes. Through pedigree analysis, it has been confirmed that the pathogenic gene of APKD is closely linked to a small satellite DNA sequence near the 3 'end of the alpha globin gene, which is 3'HVR (3' hypervariable region), which is highly polymorphic in the population. Therefore, diagnosis can be made by RFLP linkage analysis.